Tumor Cells Of Origin Research Articles

Antibody-independent capture of circulating tumor cells of non-epithelial origin with the ApoStream® system.

Circulating tumor cells (CTCs) are increasingly employed for research and clinical monitoring of cancer, though most current methods do not permit the isolation of non-epithelial tumor cells. Furthermore, CTCs isolated with antibody-dependent methods are not suitable for downstream experimental uses, including in vitro culturing and implantation in vivo. In the present study, we describe the development, validation, and transfer across laboratories of a new antibody-independent device for the enrichment of CTCs from blood samples of patients with various cancer diagnoses. The ApoStream® device uses dielectrophoresis (DEP) field-flow assist to separate non-hematopoietic cells from the peripheral blood mononuclear fraction by exposing cells in a laminar flow stream to a critical alternating current frequency. The ApoStream® device was calibrated and validated in a formal cross-laboratory protocol using 3 different cancer cell lines spanning a range of distinct phenotypes (A549, MDA-MB-231, and ASPS-1). In spike-recovery experiments, cancer cell recovery efficiencies appeared independent of spiking level and averaged between 68% and 55%, depending on the cell line. No inter-run carryover was detected in control samples. Moreover, the clinical-readiness of the device in the context of non-epithelial cancers was evaluated with blood specimens from fifteen patients with metastatic sarcoma. The ApoStream® device successfully isolated CTCs from all patients with sarcomas examined, and the phenotypic heterogeneity of the enriched cells was demonstrated by fluorescence in situ hybridization or with multiplex immunophenotyping panels. Therefore, the ApoStream® technology expands the clinical utility of CTC evaluation to mesenchymal cancers.

Dimensions of Niche-Induced Anti-Apoptosis of Therapeutically Resistant Neoplasms in Radio-Chemo-Therapy

It is proposed that apoptotic linear progression schemes do not adequately account for the emergence of non response in radio chemoresistant neoplastic lesions In such defining contributory patterns of activated and non activated apoptosis there is emerging evidence for distributional programs sustained by networks of spatial distribution Such a concept may well implicate angiogenic switches and origins of vessels and tumor cells from a resident stem cell population in the niche micro environment It is towards the realization of performance attributes of cell cycle arrest on the one hand and of proliferative dysfunctionality that the resistance and relapse of tumors is exemplified by an integrated clonogenic phenomenon of cellular derivation and end result as relapsed neoplasms

Freezing Nitrogen Ethanol Composite May be a Viable Approach for Cryotherapy of Human Giant Cell Tumor of Bone.

Liquid nitrogen has been used as adjuvant cryotherapy for treating giant cell tumor (GCT) of bone. However, the liquid phase and ultrafreezing (-196°C) properties increase the risk of damage to the adjacent tissues and may lead to perioperative complications. A novel semisolid cryogen, freezing nitrogen ethanol composite, might mitigate these shortcomings because of less-extreme freezing. We therefore wished to evaluate freezing nitrogen ethanol composite as a coolant to determine its properties in tumor cryoablation. (1) Is freezing nitrogen ethanol composite-mediated freezing effective for tumor cryoablation in an ex vivo model, and if yes, is apoptosis involved in the tumor-killing mechanism? (2) Does freezing nitrogen ethanol composite treatment block neovascularization and neoplastic progression of the grafted GCTs and is it comparable to that of liquid nitrogen in an in vivo chicken model? (3) Can use of freezing nitrogen ethanol composite as an adjuvant to curettage result in successful short-term treatment, defined as absence of GCT recurrence at a minimum of 1year in a small proof-of-concept clinical series? The cryogenic effect on bone tissue mediated by freezing nitrogen ethanol composite and liquid nitrogen was verified by thermal measurement in a time-course manner. Cryoablation on human GCT tissue was examined ex vivo for effect on morphologic features (cell shrinkage) and DNA fragmentation (apoptosis). The presumed mechanism was investigated by molecular analysis of apoptosis regulatory proteins including caspases 3, 8, and 9 and Bax/Bcl-2. Chicken chorioallantoic membrane was used as an in vivo model to evaluate the effects of freezing nitrogen ethanol composite and liquid nitrogen treatment on GCT-derived neovascularization and tumor neoplasm. A small group of patients with GCT of bone was treated by curettage and adjuvant freezing nitrogen ethanol composite cryotherapy in a proof-of-concept study. Tumor recurrence and perioperative complications were evaluated at a minimum of 19months followup (mean, 24months; range, 19-30months). Freshly prepared freezing nitrogen ethanol composite froze to -136°C and achieved -122°C isotherm across a piece of 10±0.50-mm-thick bone with a freezing rate of -34°C per minute, a temperature expected to meet clinical tumor-killing requirements. Human GCT tissues revealed histologic changes including shrinkage in morphologic features of multinucleated giant cells in the liquid nitrogen (202±45μm; p=0.006) and freezing nitrogen ethanol composite groups (169±27.4μm; p<0.001), and a decreased nucleated area of neoplastic stromal cells for the 30-second treatment. Enhanced counts of terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive cells verified the involvement of DNA fragmentation in cryoablated GCT tissues. Western blotting analysis on the expression of apoptosis regulatory proteins showed enhancement of proteocleavage-activated caspases 3, 8, and 9 and higher ratios of Bax/Bcl2 in the liquid nitrogen- and freezing nitrogen ethanol composite-treated samples. Numbers of blood vessels and human origin tumor cells also were decreased by freezing nitrogen ethanol composite and liquid nitrogen treatment in the GCT-grafted chicken chorioallantoic membrane model. Seven patients with GCT treated by curettage and adjuvant cryotherapy by use of freezing nitrogen ethanol composite preparation had no intra- or postoperative complications related to the freezing, and no recurrences during the study surveillance period. These preliminary in vitro and clinical findings suggest that freezing nitrogen ethanol composite may be an effective cryogen showing ex vivo and in vivo tumor cryoablation comparable to liquid nitrogen. The semisolid phase and proper thermal conduction might avoid some of the disadvantages of liquid nitrogen in cryotherapy, but a larger clinical study is needed to confirm these findings. Level IV, therapeutic study.

The ascites N-glycome of epithelial ovarian cancer patients

Epithelial ovarian cancer (EOC) is worldwide the sixth most lethal form of cancer occurring in women. More than one third of ovarian patients have ascites at the time of diagnosis and almost all of them have it when recurrence occurs. Although its effect on tumor cell microenvironment remains poorly understood, its presence is correlated with bad diagnosis. In previous studies, we proposed a novel glycan-based biomarker for the diagnosis of EOC, which showed an improved sensitivity and specificity at any stage of the disease and an improved discrimination between malignant and benign ovarian tumors. In this work, we report for the first time the N-glycome profiles of ascitic fluid from primary serous EOC patients and compare them with the serum N-glycomes of the same patients as well as of healthy controls. N-Glycans were digested from equivalent amount of ascites and serum from 18 EOC patients and from serum of 20 age-matched controls and measured by MALDI-TOF-MS. Ascites N-glycome showed increased antennarity, branching, sialylation and LewisX motives compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation. SignificanceMalignant ascitic fluid is the build-up of large volumes of fluid in the peritoneal cavity secondary to cancer. At least one-third of ovarian cancer patients develop ascites, a generally voluminous fluid containing cells of tumor origin, in the course of cancer and almost all when recurrence occurs. The proteome of ascites is known to be as complex as that of serum and contains high amount of proteins shed from inflammatory cells as well as from tumor cells. Although many attempts have been made to provide molecular insight into the proteomic and peptidomic content of malignant ascites, no data about the N-glycome of the ascitic fluid fraction from cancer patients has been reported to date.In this study, the N-glycosylation profile of ascites from 20 patients suffering from epithelial ovarian cancer (EOC) was analyzed by MALDI-TOF-mass spectrometry and compared to the pathologically modified N-glycan pattern obtained from serum of the same patients as well as to the pattern of serum from healthy individuals. Significant quantitative differences were observed in the ascites of EOC patients when compared to the serum of healthy subjects. The glycome of ascites shows typical features of inflammatory conditions, what was also found in the serum of patients suffering from EOC when compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation, showing that the high-volume ascites contains a higher amount of sialylated structures than the low-volume ascites.

Pathological diagnosis and differential diagnosis for primary pulmonary mucinous epithelial tumors

The cell origin of primary pulmonary mucinous epithelial tumors includes goblet cells, tracheobronchial mucous glands, the mucous cell metaplasia of ciliated and Clara cells, etc.There are benign, low-grade malignant potential and malignant tumors in this category. The benign tumors encompass mucous gland adenoma and mucinous cystadenoma. Ciliated muconodular papillary tumors are thought to be of low grade malignant potential or uncertain malignant potential neoplasm, while colloid adenocarcinoma and mucinous adenocarcinoma are malignant tumors. Most of primary pulmonary mucinous epithelial tumors are rare even extremely rare lesions. Similar morphological changes exist in the different tumors. Differential diagnosis for these entities may be challenging in pathological diagnosis on biopsies, even surgical sections. The clinicopathologic characteristics should be carefully analyzed to ensure accurate pathologic diagnosis for primary pulmonary mucinous epithelial tumors.

Is Circulating Tumor DNA (Ctdna) Use Ready For Prime Time? Applications and Challenges of Ctdna in the Era of Precision Oncology

“Liquid biopsies” have emerged as a tool to monitor genomic alterations in the peripheral blood [1]. Cell-free DNA consists of noncancerous nucleic acids and circulating tumor DNA (ctDNA). The proportion of ctDNA depends on the tumor cell of origin and stage of malignancy [2-5]. Peripheral blood biopsies can detect single nucleotide variants, indels, copy number variants, rearrangements and fusions, non-invasively, avoiding risk associated with repeat tissue biopsies. However, concerns remain with respect to ctDNA adequately reflecting tumor heterogeneity, thresholds for detection, and lack of randomized control trials to validate improved survival outcomes. As a result, further data are necessary to compare sequencing data between tissue and blood biopsies to validate clinical utility.

Immunohistochemical characterization of urethane-induced lung tumors in CB6F1-Tg rasH2 mice.

The purpose of this study was to define the histopathological characteristics of pulmonary neoplastic lesions, especially focusing on the origin of tumor cells, in urethane-treated Tg rasH2 mice. Bronchiolar-alveolar adenomas/adenocarcinomas were observed in the lungs from all of the urethane-treated animals. Immunohistochemically, these tumors showed an alveolar epithelial type II (AE2) cell phenotype demonstrating positive staining of surfactant protein C (SP-C). Cells expressing Clara cell 10 (CC10), a Clara cell marker, were also observed in a scattered manner in some tumors. Several SP-C and CC10 double-positive cells were observed in these tumors. Most of the urethane-induced pulmonary tumors were considered to have an AE2 cell phenotype, but the presence of SP-C and CC10 double positive cells in the tumors of Tg rasH2 mice suggests that some tumors arose from bronchioalveolar stem cells, which are known to express both SP-C and CC10.

P2.01-077 Serum CYFRA 21-1 and CEA Level as a Predicting Marker for Advanced Non-Small Cell Lung Cancer

Tumor markers such as CEA and CYFRA 21-1 have been shown to be effective in patients with non-small cell lung cancer as an aid in diagnosis and monitoring response to treatment. CYFRA 21-1 is a fragment of cytokeratin 19, presented in the cytoplasm of tumor cells of epithelial origin. CEA or Carcino-Embryonic Antigen is a cell surface protein. We recruited advanced non-small cell lung cancer patients who received first line treatment with standard chemotherapy between February and September 2015 in Maharaj Nakorn ChiangMai hospital. Excluded criteria were patients who had history of other active cancers and who had end stage renal disease (confounding factors to serum CYFRA measurement). Clinical data was obtained and a blood sample was collected at baseline, after start chemotherapy at cycle 2nd and at the end of treatment. Serum tumor marker levels were determined with a test kit (Roche Diagnostics Corp) using a Cobas e 411 analyzer. Primary objective outcome is correlation in dynamic change of serum CYFRA and CEA level compare to clinical response from radiologic assessment. Secondary objective outcome Secondary objective outcome is optimal cut point to predict the treatment outcome from sensitivity and specificity analysis. Multivariable logistic regression analysis was used to identify the effect of changed value of CYFRA-21. C-statistic was used to identify the optimal cut of point of changed value of CYFRA-21 to predict the outcome of treatment demonstrated by area under receiver operating characteristic curve (AuROC). The p-value < 0.05 was considered statistically significant. All statistical analysis was performed using STATA program (version 12.0). Forty patients (24 males and 16 females) were enrolled. The median age was 59.8 years. Histology subtypes were adenocarcinoma (70%), squamous cell (22.5%), large cell carcinoma (5%) and NOS(not otherwise specified) (2.5%). The treatment responses were partial response (50%), stable of disease (27.5%) and progression of disease (22.5%). The result demonstrated significant correlation between dynamic change of serum CYFRA level and clinical benefit from radiologic assessment. Mean change value of CYFRA-21 before and after treatment between clinical beneficial group (PR + SD) and PD group were -7.7±9.2 and 12.5±23.2 respectively (p<0.001). In contrast dynamic change of serum CEA did not show significant correlation.For secondary end point, at cut point of 2 ng/ml reduction of CYFRA level after treatment had the most accepted from AuROC curve. CYFRA 21-1 have capability to predict benefit of treatment from chemotherapy in non-small cell lung cancer.

Clinical Significance of Disseminated Pluripotent Tumor Cell Signature Expression in the Bone Marrow from Patients with Colorectal Cancer.

Purpose:Disseminated tumor cells (DTCs) are critically involved in tumor relapse and survival in several invasive tumors. We previously showed that the ATP-binding cassette (ABC) transporter, ABCB5, is a chemoresistance mediator expressed on specific cell subsets in colorectal cancer (CRC) and other malignancies. This study evaluated the molecular signature expression and its clinical relevance of DTCs in bone marrow from patients with colon cancer.Methods:This study included 49 consecutive patients (UICC stage I-IV) that underwent curatively intended or palliative surgery for CRC. We analyzed cells from bone marrow aspirates obtained before surgery and derived from patients that had completed minimally a 5-year follow-up. The gene expression of ABCB5 in comparison to CD133 (molecule for identifying cancer initiating cells), Lgr5 (an intestinal stem cell marker) as well as Cytokeratin (CK) 20 (terminally differentiated tumor cells of epithelial origin) in these cells was evaluated.Results:Bone marrow analysis showed differential expression between the analyzed genes. ABCB5 and Lgr5 and to lesser extent CD133 and CK20 genes were significantly expressed in the analyzed cells from bone marrow aspirates while only ABCB5 and Lgr5 were significantly negative associated with tumor progress and overall survival.Conclusion:Overexpression of ABCB5 and Lgr5 in bone marrow negatively influenced patient survival pointing to a specific chemo resistant and pluripotent cell subgroup of DTCs in the bone marrow. ABCB5 like Lgr5 positive cells seem to be involved in limited tumor related patient survival, suggesting that ABCB5- and Lgr5-positive cells may be relevant for specific clinical intervention strategies

Intensification and redistribution of protrusive activity is a feature of tumor transformation and is associated with an increase of the invasive potential of cells

The abilities of tumor cells to invade and metastasize are frequent causes of death of cancer patients. Studying the mechanisms of cell motility alterations and acquisition of enhanced metastatic potential as the result of transformation is an important aspect in current cell biology. The initial and determinant step of cell motility is the formation of active cell edge with protrusions based on the Arp2/3-dependent actin polymerization. We used three different cell systems as examples of different models of tumor transformation to study the alteration and redistribution of protrusive activity caused by transformation in fibroblasts. We analyzed relationships between detected alterations and the acquisition of increased invasive potential by cells. Active edge of untransformed fibroblasts occupies about 50% of the cell perimeter and is concentrated at the cell front. There are well pronounced stable regions at the lateral cell edges. Tumor transformation causes redistribution of protrusive activity of fibroblasts irrespective of their origin and the nature of transforming agents. The length of active edges significantly increases, up to 92% of the total perimeter in fibrosarcoma cells of tumor origin. These cells have practically no stable edges. The intensity of protrusive activity of transformed cells is also increased. Single transformed cells show a decrease in the directionality and rate of migration on 2D substrate without special stimulation. Instead, they gain the capacity to migrate in 3D and to invade matrigel. These abilities increase in parallel with the intensification of edge activity. We showed that invasive abilities are not associated with the activation of matrix metalloproteinases in the studied cell systems. Our data demonstrate that the increase of length of active edge could be considered as an additional feature of cell transformation together with the reduction of stress fiber and focal adhesions and that the excessive protrusive activity results in the development of explorative migration of tumor cells.

Live Fibular Graft for the Treatment of Long Bone Tumors in Children

Purposes: We would like to inform the characteristics of recurred osteo-fibrous dysplasia (OFD), and also the possible use of vascularized composite autograft and allografts (VCA) combined with live fibular graft (LFG) for the treatment of aggressive benign bone tumor and osteogenic sarcomas in children. Materials and Methods: We reviewed one boy with recurred OFD after LFG, and other four children with osteogenic sarcoma in long bone which was treated with LFG and VCA, and followed them for average 9 years (3 - 14 years). Survival of the LFG and VCA was estimated by the ISOLS Functional Radiologic Scoring System, but not with Kaplans-Meier’s scoring system because of a small series of case reports. Results: All succeed after surgeries initially, but the cause of recurrence of OFD was still unclear, and one girl with osteogenic sarcoma on distal femur died with skip and lung metastasis, 4 years after surgeries. Conclusions: The causes of recurred OFD are not found thru pathologic studies of our patient, but we believe the multifocal origin of tumor cells even on the adjacent soft tissues in OFD is one of the causes. The excised long bone tumor is recycled by pasteurization or autoclaving, or allograft, then LFG, and neo-adjuvant chemotherapy would be one of elective surgery for the treatment of malignant long bone tumors in children. The LFG into recycled autograft or allograft bone is difficult to perform simultaneously, but very effective to increase more earlier regenerative vascularities and also the stabilities of the dead bones in children.

Leukemic Cell Expressed CTLA-4 Suppresses T Cells Via Down-Modulation of CD80 By Trans-Endocytosis

The function of CTLA-4 on non-T cells is largely ignored and currently ill defined despite rapidly growing interest in targeting this immune checkpoint protein in several cancers. While anti-CTLA-4 therapy is proposed to work through inhibition of the immunosuppressive effect of CTLA-4 on T cells, multiple examples of non-T cell expressed CTLA-4 have been reported. These cells include tumor cells of hematological and non-hematological origin and normal B cells. In this study, we have defined a novel immune suppressive role for non-T cell, tumor expressed CTLA-4 in Chronic Lymphocytic Leukemia (CLL). We have detected by microarray that CTLA-4 is in the top 5 most differentially expressed genes between pooled samples of healthy donor normal B cells (N=6) and pooled CLL leukemic B cells (N=5). Upregulation of CTLA-4 by CLL B cells compared to normal B cells was validated by RT-qPCR and flow cytometry. CTLA-4 was predominantly intracellular (42/46 CTLA-4+) and not on the cell surface (2/48 CTLA-4+) in primary CLL samples. B cell activating factors (CD40L, PMA/Ionomycin, LPS, IL4, LPS+IL4, CD40L+IL4, CpG, and anti-IgM) could not induce surface expression of CTLA-4; however, co-culture with anti-CD3/anti-CD28 or ConA activated T cells (autologous or allogeneic) resulted in detectable CTLA-4 on the cell surface of leukemic B cells. This induction did not occur with resting T cells. This finding suggests a role for CTLA-4+ tumor cells in sites of T cell activation, such as the lymph node, a site of leukemic cell proliferation in CLL. To mechanistically study leukemic B cell expressed CTLA-4, we generated CLL-derived Mec1 and OSU-CLL that inducibly express CTLA-4 upon doxycycline (dox) treatment. Mec1 and OSU-CLL cells highly express the ligands for CTLA-4, CD80 and CD86. Dox-induction of CTLA-4 resulted in decreased expression of Mec1 and OSU-CLL expressed CD80, a critical T cell co-stimulatory protein (N=3). Blockade of CTLA-4 using the anti-CTLA-4 therapeutic antibody, Ipilimumab, could restore CD80 on Mec1 and OSU-CLL cells (N=3). Because T cell-expressed CTLA-4 has been previously shown by others to down-modulate CD80 via trans-endocytosis, we co-cultured CTLA-4+ Mec1 and CTLA-4+ primary CLL cells with stably transfected CD80-GFP or CD86-GFP Hek293 cell lines to assess uptake of CD80/CD86 into CTLA-4 expressing tumor cells as the mechanism of CD80 down-modulation. Transfer of CD80-GFP and CD86-GFP was detected by flow cytometry in primary CLL cells and the Mec1 cell line, consistent with the ability of T cell expressed CTLA-4 to trans-endocytose CD80 and CD86. Furthermore, uptake of CD80-GFP or CD86-GFP by primary tumor cells was CTLA-4 dependent, demonstrated by inhibition of GFP uptake in the presence of Ipilimumab. Following determination of decreased CD80, we found that co-culture of primary T cells with Mec1 CTLA-4+ cells resulted in decreased IL2 production measured by Cytokine Bead Array. The loss of IL2 signified decreased co-stimulation as a result of tumor expressed CTLA-4. Studies are ongoing regarding dependence on CD80 or CD86. A minor subset of T cells, Tregs, are known to exert profound immunosuppressive effects through their expression of CTLA-4. Due to our results, tumor expressed CTLA-4 has an overlapping function with Treg CTLA-4, and it is imperative that we define the immunosuppressive effects as, in patients, the leukemic cells may comprise a much larger proportion of white blood cells than T cells. Efforts are now underway to address the effect of tumor expressed CTLA-4 in suppressing anti-tumor immunity in vivo utilizing a novel mouse model. Suppression of T cells by tumor expressed CTLA-4 is a novel finding that is broadly applicable to fields within and outside of cancer research as the pathway and mechanism described here are potentially applicable to CTLA-4 in diverse disease contexts and to the general biology of CTLA-4.[Funding: This work was supported by P01 CA95426. PD received the Pelotonia Graduate Fellowship. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the Pelotonia Fellowship Program] DisclosuresJones:AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Ion channels in control of pancreatic stellate cell migration.

Pancreatic stellate cells (PSCs) play a critical role in the progression of pancreatic ductal adenocarcinoma (PDAC). Once activated, PSCs support proliferation and metastasis of carcinoma cells. PSCs even co-metastasise with carcinoma cells. This requires the ability of PSCs to migrate. In recent years, it has been established that almost all “hallmarks of cancer” such as proliferation or migration/invasion also rely on the expression and function of ion channels. So far, there is only very limited information about the function of ion channels in PSCs. Yet, there is growing evidence that ion channels in stromal cells also contribute to tumor progression. Here we investigated the function of KCa3.1 channels in PSCs. KCa3.1 channels are also found in many tumor cells of different origin. We revealed the functional expression of KCa3.1 channels by means of Western blot, immunofluorescence and patch clamp analysis. The impact of KCa3.1 channel activity on PSC function was determined with live-cell imaging and by measuring the intracellular Ca2+ concentration ([Ca2+]i). KCa3.1 channel blockade or knockout prevents the stimulation of PSC migration and chemotaxis by reducing the [Ca2+]i and calpain activity. KCa3.1 channels functionally cooperate with TRPC3 channels that are upregulated in PDAC stroma. Knockdown of TRPC3 channels largely abolishes the impact of KCa3.1 channels on PSC migration. In summary, our results clearly show that ion channels are crucial players in PSC physiology and pathophysiology.

Abstract A023: Inducing neoantigens in disseminated tumor lesions to enhance their susceptibility to PD-1 blockade therapy

Abstract Correlation between “clonal” neoantigen burden (neoantigens generated early during cancer development and therefore represented at high frequency in all tumor lesions) and responsiveness to checkpoint blockade therapy has underscored the importance of neoantigens in promoting tumor immunogenicity, and has spurred a broad effort to develop methods to identify neoantigens for use in vaccination protocols. Yet, the majority of patients do not express, or express low numbers of, clonal neoantigens, and consequently will be less likely to benefit from checkpoint blockade and other forms of immune potentiating therapies. Here we describe several strategies to generate de novo neoantigens in the patient' disseminated tumor lesions and show that in murine tumor models they potentiate checkpoint blockade therapy. We have previously shown that oligonucleotide aptamer-targeted siRNA inhibition of the Nonsense-mediated mRNA (NMD) process in tumor cells in situ results in the induction of neoatigens and inhibition of tumor growth (Pastor et al., Nature, 2010, 465:227). We now demonstrate that tumor-targeted NMD inhibition potentiates PD-1 blockade therapy. A general concern and potential limitation of the NMD approach is that a significant proportion of NMD inhibition-induced neoantigens are generated as a result of random mutations or aberrant splicing and hence will not be represented at high frequency in all the tumor lesions of the patient. To that end, we are exploring alternative strategies to generate clonal neoantigens by targeting key components of antigen presentation pathways, specifically the TAP transporter, ERAAP peptidase, and Invariant chain, the latter to generate class II-restricted neoantigens. Studies have shown that downregulation of these products, using genetic means or antisense RNA, not only inhibits the canonical antigen presentation pathway but also upregulates alternative pathways that present new, otherwise silent or subdominant, epitopes. Since such epitopes are not generated as a result of random mutations they are more likely to represent “clonal” neoepitopes generated in every cell in which the target was downregulated. Here we are developing clinically feasible broadly applicable approaches to inhibit the aforementioned mediators of antigen presentation using corresponding siRNAs targeted to tumor cells in situ by conjugation to a nucleolin-binding aptamer. Nucleolin, a nucleolar product, is translocated to the surface the majority of tumor cells of both human and murine origin and thereby serves as a broad, if not universal, target to deliver therapeutic cargo to the disseminated tumor cells of the patient. We show that nucleolin aptamer-targeted downregulation of ERAAP or Invariant chain in tumor-bearing mice inhibits tumor growth and potentiates PD-1 blockade therapy. Ongoing studies explore the combinatorial use of neoantigen induction methods and combination of neoantigen induction with immune potentiating strategies including but not limited to checkpoint blockade. Citation Format: Greta Garrido Hidalgo, Agata Levay, Alexey Berezhnoy, Brett Schrand, Eli Gilboa. Inducing neoantigens in disseminated tumor lesions to enhance their susceptibility to PD-1 blockade therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A023.

The clinical value of pleural effusion by liquid-based cytology combined with cell block and immunohistochemical to prompt the origin of tumor cells

Objective To investigate application values of Liquid-based cytology(TCT), cell block and immunohistochemical on estimating sources of malignant tumor cells in hydrothorax. Methods Collect 113 cases of Pleural effusion by Liquid-based cytology that are tentatively diagnosed as malignant tumor or suspected malignancies from January 2013 to June 2016 in Yuncheng Central Hospital. Through centrifuging to collect sediment cells and make paraffin block, 99 cases are selected for immunohistochemical examinations, through the CK7,CK20 and villin immunohistochemical staining determine the direction of the source of tumor, then targeted choose the TTF-1,NapsinA,CK5/6,p63,CR,Syn,CgA,CA125,GCDFP- 15,ER,PR and CDX-2 antibody to immunohistochemical staining. Results Among 94 cases of adenocarcinomas by TCT, 92 cases are still diagnosed as adenocarcinomas by immunohistochemistry. In addition, tumours sources of 92 cases are as following: 70 cases are from lung, 2 cases are from breast, 6 cases are from ovary, 8 cases are from alimentary canal, 1 case is from peritoneal serouspapillary carcinoma, and 5 cases are unknown sources. The other 2 cases are definitely diagnosed as reactive mesothelial cells and tumors of mesothelium respectively. The same diagnostic results of squamous-cell carcinoma and small cell carcinoma by TCT, cell block and Immunohistochemistry are 2 cases and 3 case respectively. The sensitivity and idiosyncrasy of Immunohistochemistry on determining the direction of tumours sources are 94.6% and 100% respectively, which are better than histological examination. Conclusions Making hydrothorax into cell blocks for immunohistochemical examination not only save tumor cells in hydrothorax, but also gathering and collecting tumor cells by centrifuge improves positive detection rate of cellular pathology and contributes to differential diagnosis of tumour cell, analysis of tissues sources, gene mutations detection and molecular targeted therapy. Therefore, it is significant to prolong patients'lives, lessen their pain, improve living quality and clinical values. Key words: Liquid-based cytology; Cell block; Immunohistochemica; Tumor cell origin

Worse or better?—Cirrhosis with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) accounts for about 90% of all malignant tumors of liver, ranking fifth in the worldwide incidence of malignant tumors and the third in fatality. More and more evidences suggest that cancer is a metabolic-related disease. From the analysis of recent clinical research data, we found that as the severity of the cirrhosis aggravated, patients with HCC and end-stage liver cirrhosis had a flat energy metabolism which was better than it in patients with simple end-stage liver cirrhosis. Based on these clinical phenomenon, the major aim of this study is to present a new hypothesis: “compensated liver function mechanism” for patients with HCC and liver cirrhosis, cancer cells may play a role to compensate liver function. In this study, we elaborated relevant content about this novel standpoint combined with tumor energy metabolism reprogramming mechanism and tumor cell origin as well as cell exchange mechanism.

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells.

The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype.

Abstract 2526: Regulation of angiogenic factors in glioblastoma progression

Abstract Background &amp; Purpose: Glioblastoma is the most malignant primary brain tumor in humans and has a very low survival rate. The aggressiveness of this cancer can be attributed to the presence of glioblastoma stem cells (GSC), which are highly invasive and therapy resistant. Increased aggressiveness of GSCs is attributed to the release of angiogenic factors, which promotes the formation of new blood vessels that enhances tumor cell survival and proliferation. The neovascularization also provides a scaffold for tumor cells to migrate, enabling metastasis. Therefore, identifying the altered expression of angiogenic factors is of clinical importance. In this study, we examined the changes in the expression of pro-angiogenic factors in GSCs as compared to the original tumor from which they were derived. We also determined if differentiation of GSC towards a less invasive phenotype decreased the levels of these pro-angiogenic factors. Methods: GBM tumor cells were grown in complete media containing 10% Fetal Calf Serum (FCS) and GSCs were grown in media containing B27, heparin, EGF, and FGF. GSCs were differentiated by growing them in media containing 10% FCS and B27 with vitamin A, for about one week. We examined the expression of a panel of pro-angiogenic factors in GSCs (33-GSC) and in the original tumor cells (33-T) from which GSCs were derived. We then examined if differentiation of 33-GSC decreased the levels of these pro-angiogenic factors. Changes in protein levels were evaluated by immunofluorescence staining. The mRNA levels were quantitated using real time Q-PCR. Results: Immunofluorescence staining showed that the GSCs express higher levels of pro-angiogenic factors examined (11 out of 16). GSCs were successfully differentiated into astrocytes as indicated by morphology and GFAP expression. The differentiation of GSCs into astrocytes was sufficient to cause decreased expression of VEGF and c-MYC. Our data showed 27 fold increase in VEGF mRNA levels, 63 fold increase in MMP2 mRNA levels, 11 fold increase in MMP9 mRNA levels, and 2 fold increase in c-MYC mRNA levels in GSCs compared to the tumor line, indicating the regulation of these pro-angiogenic factors at transcriptional level. Conclusions: Increased expression of pro-angiogenic factors in GSCs may render these cells to be more aggressive and invasive, and suggests that differential expression of angiogenic factors in GSC cells may play an important role in the GBM progression, and therapy resistance. Citation Format: Carolina Larrain, Umadevi V. Wesley, Paul Clark, John S. Kuo, Robert Dempsey. Regulation of angiogenic factors in glioblastoma progression. [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 2526.

Abstract 633: Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer

Abstract Background: Patient derived xenografts (PDX) are regarded as the gold-standard for translational preclinical cancer research due to their preservation of tumor heterogeneity and propagation in vivo. Similarly, grafting human tumor cells of a particular tissue origin into the corresponding orthotopic context enhances translational accuracy; with recapitulation of tumor::microenvironment biology, as well as more accurate tumor progression kinetics and metastasis, a s well as for systemic delivery of therapies as compared to traditional subcutaneous xenografts. Bioluminescent luciferase-reporters into traditional cancer cell lines has allowed for in-life imaging of tumor growth kinetics in real-time, within obfuscated internal organs, improving experimental precision. We have reported on the development and validation of bioluminescent pancreatic cancer and breast cancer PDX in clinically recapitulative orthotopic models. Here, we characterize these bioluminescent PDX by gene-expression profiling; comparing orthotopic and ectopically propagated bioluminescent PDX to their non-transduced parental PDX counterparts. Methods: Pancreatic (PANx-005-Luc) or metastatic breast (HBCx-14-Luc) PDXs stably transduced with lentiviral luciferase were dissociated and implanted orthotopically into pancreas or mammary fat pad respectively or subcutaneously into the flank of NOD scid gamma (NSG) mice. Likewise, the parental reporter-free PANx-005 or HBCx-14 PDX were implanted subcutaneously into NSG mice. For luciferase-expressing PDX implanted orthotopically, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). For the PANx-005-Luc, orthotopic tumors were harvested at a mean tumor radiance (TR) of 2.53 × 106 + 1.63 × 106 ph/s, while ectopic luciferase-transduced and parental tumors were harvested at a mean tumor volume of 530 + 337mm3. For the HBCx-14-Luc, orthotopic tumors were harvested individually when TR reached 6.5 × 109 ph/s while ectopic luciferase-transduced or parental tumors were harvested when tumor volumes reached 1500 + 362.5mm3. Tumor samples were fixed in 10% neutral buffered formalin and embedded in paraffin-blocks for histological analysis. RNA was extracted from all tumors and full-coverage human genome gene-expression profiling was performed. Major differences observed in microarray data was validated via quantitative biochemical assays. Results: Stable expression of lentiviral luciferase did not result in significant gene-expression changes to PDX lines, while site of implantation altered some gene-expression characteristics of the PDX. Conclusions: The introduction of an integrating stable bioluminescent reporter does not significantly alter the gene-expression characteristics of PANx-005 or HBCx-14 PDX. Site of implantation plays a role in gene-expression of PDX tumor models. Citation Format: Benjamin G. Cuiffo, Olivier Deas, Ingrid Rankin, Gregory D. Lyng, Stephano Cairo, Katie Pedrick, Alexandra Kury, Enora Le Ven, Jean-Gabriel Judde, Stephen T. Sonis. Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer. [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 633.