Primary Cancer Cell Lines Research Articles

Cell-cell and cell-substratum contacts in the regulation of MAPK and Akt signalling: Importance in therapy, biopharmacy and bioproduction

The use of cultured cells as a tool for research, precision medicine, biopharmacy, and biomanufacturing is constantly increasing. In parallel, the role of cell-cell and cell-substratum contacts in cell functioning is increasingly validated. Adhesion signalling plays a key role here. The activity of cell fate-regulating signalling molecules is an important factor in determining cell behaviour, as well as their response to treatment, depending on cell phenotypic status and location in the body. Three cellular state models (adherent, single cells in suspension, and aggregated cells) were compared for cell signalling, including focal adhesion (FAK), mitogen-activated (MAPK), as well as Akt protein kinases, and transcription factor cJun, by using lung adenocarcinoma A549, muscle-derived stem Myo, as well as primary lung cancer cell lines. Survival of both A549 and Myo cells was dependent on kinases Akt and ERK in detached conditions. Intercellular contacts in aggregates promoted activation of Akt and ERK, and cell survival. Loss of contacts with the substrate increased phosphorylation of MAP kinases JNK and p38, while decreased Akt phosphorylation by processes involving FAK. Unexpectedly, detachment increased phosphorylation of antiapoptotic kinase ERK in A549, while in Myo stem cells ERK phosphorylation was downregulated. JNK target transcription factor cJun protein level was markedly diminished by contacts between cells possibly involving mechanism of proteasomal degradation. Furthermore, studies revealed the opposite dependence of molecules of the same signalling pathway – phospho-cJun and phospho-JNK – on cell culture density. Differences in ERK activation under detachment conditions indicate that targeting of prosurvival kinases during anoikis should be different in different cells. Moreover, the outcome of JNK activation in cells may depend on the amount of cJun, which is determined by cell-cell contacts.

Creation and Maintenance of a Living Biobank - How We Do It.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

Establishment and characterization of 38 novel patient-derived primary cancer cell lines using multi-region sampling revealing intra-tumor heterogeneity of gallbladder carcinoma

Gallbladder carcinoma (GBC) is a lethal biliary tract malignant neoplasm. Patient-derived primary cancer cell lines (PDPCs) are appropriate models to explore biological characteristics and potential therapeutics; however, there is a lack of PDPCs in GBC. In this study, we aimed to establish and characterize the GBC PDPCs, and further investigated the intra-tumor heterogeneity (ITH). Multi-region sampling (3–9 regions) of the operable tumor tissue samples was used to establish PDPCs. Short tandem repeat genotyping for cell authentication and karyotyping was performed, followed by whole-exome sequencing and RNA sequencing to assess the ITH at the genetic and transcriptional levels, respectively. Thirty-eight PDPCs were successfully established from seven GBC patients and characterized. ITH was observed with a median of 38.3% mutations being heterogeneous (range, 26.6–59.4%) across all patients. Similar with other tumor types, TP53 mutations were always truncal. In addition, there were three genes, KMT2C, CDKN2A, and ARID1A, with truncal mutations in at least two patients. A median of 370 differentially expressed genes (DEGs) was identified per patient. Distinct expression patterns were observed between major histocompatibility complex (MHC) class I and II genes. We found the expression of MHC class II genes in the PDPC samples was closely regulated by CIITA, while that of MHC class I genes were not correlated with CIITA expression. The PDPCs established from GBC patients can serve as novel in vitro models to identify the ITH, which may pave a crucial molecular foundation for enhanced understanding of tumorigenesis and progression.

LMNB2 promotes the progression of colorectal cancer by silencing p21 expression

Colorectal cancer is the second common cause of death worldwide. Lamin B2 (LMNB2) is involved in chromatin remodeling and the rupture and reorganization of nuclear membrane during mitosis, which is necessary for eukaryotic cell proliferation. However, the role of LMNB2 in colorectal cancer (CRC) is poorly understood. This study explored the biological functions of LMNB2 in the progression of colorectal cancer and explored the possible molecular mechanisms. We found that LMNB2 was significantly upregulated in primary colorectal cancer tissues and cell lines, compared with paired non-cancerous tissues and normal colorectal epithelium. The high expression of LMNB2 in colorectal cancer tissues is significantly related to the clinicopathological characteristics of the patients and the shorter overall and disease-free cumulative survival. Functional analysis, including CCK8 cell proliferation test, EdU proliferation test, colony formation analysis, nude mouse xenograft, cell cycle, and apoptosis analysis showed that LMNB2 significantly promotes cell proliferation by promoting cell cycle progression in vivo and in vitro. In addition, gene set enrichment analysis, luciferase report analysis, and CHIP analysis showed that LMNB2 promotes cell proliferation by regulating the p21 promoter, whereas LMNB2 has no effect on cell apoptosis. In summary, these findings not only indicate that LMNB2 promotes the proliferation of colorectal cancer by regulating p21-mediated cell cycle progression, but also suggest the potential value of LMNB2 as a clinical prognostic marker and molecular therapy target.

Pharmaceutical immunoglobulin G impairs anti-carcinoma activity of oxaliplatin in colon cancer cells

BackgroundRecent evidence proves that intravenous human immunoglobulin G (IgG) can impair cancer cell viability. However, no study evaluated whether IgG application benefits cancer patients receiving chemotherapeutics.MethodsInfluence of pharmaceutical-grade human IgG on the viability of a series of patient-derived colon cancer cell lines with and without chemotherapeutic intervention was determined. Cell death was analysed flow cytometrically. In addition, the influence of oxaliplatin and IgG on the ERK1/2-signalling pathway was evaluated by western blots.ResultsWe evaluated the effects of pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, in combination with chemotherapeutics. We did not observe any significant effects of IgG on tumour cell viability directly; however, human IgG significantly impaired the anti-tumoral effects of oxaliplatin. Primary cancer cell lines express IgG receptors and accumulate human IgG intracellularly. Moreover, while oxaliplatin induced the activation of ERK1/2, the pharmaceutical IgG inhibited ERK1/2 activity.ConclusionsThe present study demonstrates that pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, can impair the anti-carcinoma activity of oxaliplatin. These data strongly suggest that therapeutic IgG as co-medication might have harmful side effects in cancer patients. The clinical significance of these preclinical observations absolutely advises further preclinical, as well as epidemiological and clinical research.

Polymer Pro-Drug Nanoparticles for Sustained Release of Cytotoxic Drugs Evaluated in Patient-Derived Glioblastoma Cell Lines and In Situ Gelling Formulations.

Glioblastoma (GBM) is the most common, malignant and aggressive brain tumour in adults. Despite the use of multimodal treatments, involving surgery, followed by concomitant radiotherapy and chemotherapy, the median survival for patients remains less than 15 months from diagnosis. Low penetration of drugs across the blood-brain barrier (BBB) is a dose-limiting factor for systemic GBM therapies, and as a result, post-surgical intracranial drug delivery strategies are being developed to ensure local delivery of drugs within the brain. Here we describe the effects of PEGylated poly(lactide)-poly(carbonate)-doxorubicin (DOX) nanoparticles (NPs) on the metabolic activity of primary cancer cell lines derived from adult patients following neurosurgical resection, and the commercially available GBM cell line, U87. The results showed that non-drug-loaded NPs were well tolerated at concentrations of up to 100 µg/mL while tumour cell-killing effects were observed for the DOX-NPs at the same concentrations. Further experiments evaluated the release of DOX from polymer-DOX conjugate NPs when incorporated in a thermosensitive in situ gelling poly(DL-lactic-co-glycolic acid) and poly(ethylene glycol) (PLGA/PEG) matrix paste, in order to simulate the clinical setting of a locally injected formulation for GBM following surgical tumour resection. These assays demonstrated drug release from the polymer pro-drugs, when in PLGA/PEG matrices of two formulations, over clinically relevant time scales. These findings encourage future in vivo assessment of the potential capability of polymer–drug conjugate NPs to penetrate brain parenchyma efficaciously, when released from existing interstitial delivery systems.

RNA binding protein PUM1 promotes colon cancer cell proliferation and migration

Colon cancer is the third leading cause of death worldwide and sixth in India, where it is the cause of 5.8% of the total deaths. Pumilio-1 (PUM1) is an RNA binding protein whose regulatory role is by binding to the consensus 5′UGUANAUA3′ sequence on the 3′UTR of the mRNA targets and post-transcriptionally repressing their expression. This study is the first of its kind to report the expression or function of PUM1 in colon cancer. We found that PUM1 mRNA expression is high in primary and metastatic colon cancer cell lines when compared to the normal colon cell line. Immunohistochemistry analysis showed similar trend wherein compared to the normal colon tissue, PUM1 was found to be overexpressed in both adenocarcinoma and in metastatic carcinoma. This confirms the role of PUM1 in colon cancer progression. PUM1 overexpression study in HCT116 revealed that cells transfected with PUM1 plasmid show an increased rate of proliferation, migration and colony formation. Overexpressing PUM1 increases the number and size of spheroids indicating the role of PUM1 in maintaining cancer stem cells. Overall, this is the first study that has shown the role of PUM1 in colon cancer development.

Pten-NOLC1 fusion promotes cancers involving MET and EGFR signalings

Inactivation of Pten gene through deletions and mutations leading to excessive pro-growth signaling pathway activations frequently occurs in cancers. Here, we report a Pten derived pro-cancer growth gene fusion Pten-NOLC1 originated from a chr10 genome rearrangement and identified through a transcriptome sequencing analysis of human cancers. Pten-NOLC1 fusion is present in primary human cancer samples and cancer cell lines from different organs. The product of Pten-NOLC1 is a nuclear protein that interacts and activates promoters of EGFR, c-MET, and their signaling molecules. Pten-NOLC1 promotes cancer proliferation, growth, invasion, and metastasis, and reduces the survival of animals xenografted with Pten-NOLC1-expressing cancer cells. Genomic disruption of Pten-NOLC1 induces cancer cell death, while genomic integration of this fusion gene into the liver coupled with somatic Pten deletion produces spontaneous liver cancers in mice. Our studies indicate that Pten-NOLC1 gene fusion is a driver for human cancers.

Authentication of Primary Murine Cell Lines by a Microfluidics-Based Lab-On-Chip System.

The reliable authentication of cell lines is a prerequisite for the reproducibility and replicability of experiments. A common method of cell line authentication is the fragment length analysis (FLA) of short-tandem repeats (STR) by capillary electrophoresis. However, this technique is not always accessible and is often costly. Using a microfluidic electrophoresis system, we analyzed the quality and integrity of different murine cell lines by STR profiling. As a proof of concept, we isolated and immortalized hematopoietic progenitor cells (HPC) of various genotypes through retroviral transduction of the fusion of the estrogen receptor hormone-binding domain with the coding sequence of HoxB8. Cell lines were maintained in the HPC state with Flt3 ligand (FL) and estrogen treatment and could be characterized upon differentiation. In a validation cohort, we applied this technique on primary mutant Kras-driven pancreatic cancer cell lines, which again allowed for clear discrimination. In summary, our study provides evidence that FLA of STR-amplicons by microfluidic electrophoresis allows for stringent quality control and the tracking of cross-contaminations in both genetically stable HPC lines and cancer cell lines, making it a simple and cost-efficient alternative to traditional capillary electrophoresis.

Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC, also known as pancreatic cancer) is one of the deadliest tumor types, characterized by poor prognosis, profound chemoresistance and overall low survival. Gemcitabine remains the standard of care for all stages of PDAC, however, with poor clinical benefits which is considered to be due to reduced drug availability in tumor cells. Gemcitabine-induced cytotoxicity depends upon sufficient drug uptake followed by intracellular activation. Pancreatic stellate cells (PSCs), a major stromal component of PDAC, were recently reported to scavenge active metabolites of gemcitabine, thereby making it unavailable for cancer cells. Gemcitabine uptake and processing in both tumor cells and PSCs, as well as expression analysis of its molecular metabolic regulators, was investigated in this study. We observed heterogeneous gemcitabine-induced cytotoxicity in different pancreatic cancer cells whereas it was absent in PSCs. The gemcitabine-induced cytotoxicity in pancreatic cancer cells was driven by differential expression of its molecular regulators.Gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) is attributed to cancer cell-intrinsic drug processing and the impact of the tumor microenvironment, especially pancreatic stellate cells (PSCs). This study uses human PDAC-derived paired primary cancer cells (PCCs) and PSCs from four different tumors, and the PDAC cell lines BxPC-3, Mia PaCa-2, and Panc-1, to assess the fate of gemcitabine by measuring its cellular uptake, cytotoxicity, and LC-MS/MS-based metabolite analysis. Expression analysis and siRNA-mediated knockdown of key regulators of gemcitabine (hENT1, CDA, DCK, NT5C1A) was performed. Compared to PSCs, both the paired primary PCCs and cancer cell lines showed gemcitabine-induced dose-dependent cytotoxicity, high uptake, as well as high and variable intracellular levels of gemcitabine metabolites. PSCs were gemcitabine-resistant and demonstrated significantly lower drug uptake, which was not influenced by co-culturing with their paired PCCs. Expression of key gemcitabine regulators was variable, but overall strong in the cancer cells and significantly lower or undetectable in PSCs. In cancer cells, hENT1 inhibition significantly downregulated gemcitabine uptake and cytotoxicity, whereas DCK knockdown reduced cytotoxicity. In conclusion, heterogeneity in gemcitabine processing among different pancreatic cancer cells and stellate cells results from the differential expression of molecular regulators which determines the effect of gemcitabine.

Immortalization of Salivary Gland Epithelial Cells of Xerostomic Patients: Establishment and Characterization of Novel Cell Lines.

Primary Sjögren’s Syndrome (pSS) is an autoimmune disease mainly affecting salivary and lacrimal glands. Previous pSS studies have relied on primary cell culture models or cancer cell lines with limited relevance to the disease. Our objective was to generate and characterize immortalized salivary gland epithelial cells (iSGECs) derived from labial salivary gland (LSG) biopsies of pSS patients (focus score > 1) and non-Sjögren’s Syndrome (nSS) xerostomic (i.e., sicca) female patients. To characterize iSGECs (n = 3), mRNA expression of specific epithelial and acinar cell markers was quantified by qRT-PCR. Protein expression of characterization markers was determined by immunocytochemistry and Western blot. Secretion of α-amylase by iSGECs was confirmed through colorimetric activity assay. Spheroid formation and associated alterations in expression markers were determined using matrigel-coated cell culture plates. Consistent mRNA and protein expressions of both epithelial and pro-acinar cell markers were observed in all three iSGEC lines. When cultured on matrigel medium, iSGECs formed spheroids, secreted α-amylase after β-adrenergic stimulation, and expressed multiple acinar cell markers at late passages. One iSGEC line retained adequate cell morphology without a loss of SV40Lt expression and proliferation potential after over 100 passages. In conclusion, our established iSGEC lines represent a viable model for salivary research due to their passaging capacity and maintenance of pro-acinar cell characteristics.

Mining a human transcriptome database for chemical modulators of NRF2.

Nuclear factor erythroid-2 related factor 2 (NRF2) encoded by the NFE2L2 gene is a transcription factor critical for protecting cells from chemically-induced oxidative stress. We developed computational procedures to identify chemical modulators of NRF2 in a large database of human microarray data. A gene expression biomarker was built from statistically-filtered gene lists derived from microarray experiments in primary human hepatocytes and cancer cell lines exposed to NRF2-activating chemicals (oltipraz, sulforaphane, CDDO-Im) or in which the NRF2 suppressor Keap1 was knocked down by siRNA. Directionally consistent biomarker genes were further filtered for those dependent on NRF2 using a microarray dataset from cells after NFE2L2 siRNA knockdown. The resulting 143-gene biomarker was evaluated as a predictive tool using the correlation-based Running Fisher algorithm. Using 59 gene expression comparisons from chemically-treated cells with known NRF2 activating potential, the biomarker gave a balanced accuracy of 93%. The biomarker was comprised of many well-known NRF2 target genes (AKR1B10, AKR1C1, NQO1, TXNRD1, SRXN1, GCLC, GCLM), 69% of which were found to be bound directly by NRF2 using ChIP-Seq. NRF2 activity was assessed across ~9840 microarray comparisons from ~1460 studies examining the effects of ~2260 chemicals in human cell lines. A total of 260 and 43 chemicals were found to activate or suppress NRF2, respectively, most of which have not been previously reported to modulate NRF2 activity. Using a NRF2-responsive reporter gene in HepG2 cells, we confirmed the activity of a set of chemicals predicted using the biomarker. The biomarker will be useful for future gene expression screening studies of environmentally-relevant chemicals.

Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition.

Simple SummaryThe aim of this study was to gain a better understanding of cancer stem cells, which are a small subpopulation of tumor cells with high plasticity driving tumor growth and metastasis. Here we isolated two novel colorectal cancer cell lines originating from a rectal neuroendocrine carcinoma and a colorectal adenocarcinoma, depicting stem-like properties. These in vitro models offer the possibility to evaluate pathophysiological mechanisms in order to develop tailored therapeutic strategies for distinct colorectal malignancies. Investigations revealed gene copy number gain of the N-myc proto-oncogene for both. Accordingly, inhibition of the protein–protein interaction of myc and N-myc proto-oncogenes with the myc-associated factor X utilizing small molecule KJ-Pyr-9, exhibited a significant reduction in survival of both cell lines by the induction of apoptosis. Consequently, the blockage of these interactions may serve as a possible treatment strategy for colorectal cancer cell lines with gene copy number gain of the N-myc proto-oncogene.Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial–mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.

Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

BackgroundEndometrial cancer, one of the most common malignant tumors, is a serious threat to women’s health. Endometrial hyperplasia is a precursor of endometrial cancer. S100 calcium binding protein P (S100P) has been found to play important roles in many types of cancer. The present study aimed to investigate the expression of S100P in endometrial cancer and its precursor lesions, and to explore the possible mechanisms.MethodsWe collected paraffin sections of normal endometrium, simple and complex non-atypical hyperplasia, atypical hyperplasia, and endometrioid carcinoma. The expression of S100P in endometrial cancer and its precancerous lesions was observed using immunohistochemistry. We also cultured primary endometrial cells and endometrial cancer cell lines (Ishikawa and RL95–2), and observed the expression of S100P in these cells. Laser confocal microscopy was used to observe the co-localization of S100P and its interacting protein Ezrin in RL95–2 cells. We employed lentiviruses to knockdown and overexpress S100P and then detected the F-actin distribution and cell invasion using phalloidin staining and Transwell assays.ResultsThere was a gradual increase in the S100P signal as the disease progressed from normal endometrium and simple non-atypical hyperplasia, to complex non-atypical hyperplasia, atypical hyperplasia, and then to endometrial cancer. S100P was mainly distributed in the cytoplasm and co-localized with Ezrin in endometrial cancer cells. After knocking down S100P, F-actin aggregated in the nucleus or to the local cell membrane. Furthermore, knockdown of S100P in Ishikawa cells decreased their cell invasion capability. Meanwhile, S100P overexpression in endometrial stromal cells increased cell invasion.ConclusionsThese data suggested that S100P might be involved in the occurrence and development of endometrial cancer via interaction with Ezrin and re-organization of F-actin to promote cell invasion.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Characterization and comparison of genomic profiles between primary cancer cell lines and parent atypical meningioma tumors

BackgroundMeningiomas are the second most common primary tumors of the central nervous system. However, there is a paucity of data on meningioma biology due to the lack of suitable preclinical in vitro and in vivo models. In this study, we report the establishment and characterization of patient-derived, spontaneously immortalized cancer cell lines derived from World Health Organization (WHO) grade I and atypical WHO grade II meningiomas.MethodsWe evaluated high-resolution 3T MRI neuroimaging findings in meningioma patients which were followed by histological analysis. RT-qPCR and immunostaining analyses were performed to determine the expression levels of meningioma-related factors. Additionally, flow cytometry and sorting assays were conducted to investigate and isolate the CD133 and CD44 positive cells from primary atypical meningioma cells. Further, we compared the gene expression profiles of meningiomas and cell lines derived from them by performing whole-exome sequencing of the blood and tumor samples from the patients, and the primary cancer cell lines established from the meningioma tumor.ResultsOur results were consistent with earlier studies that reported mutations in NF2, SMO, and AKT1 genes in atypical meningiomas, and we also observed mutations in MYBL2, a gene that was recently discovered. Significantly, the genomic signature was consistent between the atypical meningioma cancer cell lines and the tumor and blood samples from the patient.ConclusionOur results lead us to conclude that established meningioma cell lines with a genomic signature identical to tumors might be a valuable tool for understanding meningioma tumor biology, and for screening therapeutic agents to treat recurrent meningiomas.

Exosomes Derived from the Human Primary Colorectal Cancer Cell Line SW480 Orchestrate Fibroblast‐Led Cancer Invasion

In localized tumors, basement membrane (BM) prevents invasive outgrowth of tumor cells into surrounding tissues. When carcinomas become invasive, cancer cells either degrade BM or reprogram stromal fibroblasts to breach BM barrier and lead invasion of cancer cells into surrounding tissues in a process called fibroblast-led invasion. However, tumor-derived factors orchestrating fibroblast-led invasion remain poorly understood. Here it is shown that although early-stage primary colorectal adenocarcinoma (SW480) cells are themselves unable to invade Matrigel matrix, they secrete exosomes that reprogram normal fibroblasts to acquire de novo capacity to invade matrix and lead invasion of SW480 cells. Strikingly, cancer cells follow leading fibroblasts as collective epithelial-clusters, thereby circumventing need for epithelial to mesenchymal transition, a key event associated with invasion. Moreover, acquisition of pro-invasive phenotype by fibroblasts treated with SW480-derived exosomes relied on exosome-mediated MAPK pathway activation. Mass spectrometry-based protein profiling reveals that cancer exosomes upregulate fibroblasts proteins implicated in focal adhesion (ITGA2/A6/AV, ITGB1/B4/B5, EGFR, CRK), regulators of actin cytoskeleton (RAC1, ARF1, ARPC3, CYFIP1, NCKAP1, ICAM1, ERM complex), and signalling pathways (MAPK, Rap1, RAC1, Ras) important in pro-invasive remodeling of extracellular matrix. Blocking tumor exosome-mediated signaling to fibroblasts therefore represents an attractive therapeutic strategy in restraining tumors by perturbing stroma-driven invasive outgrowth.

Microenvironmental Th9 and Th17 lymphocytes induce metastatic spreading in lung cancer.

Immune microenvironment plays a critical role in lung cancer control versus progression and metastasis. In this investigation, we explored the effect of tumor-infiltrating lymphocyte subpopulations on lung cancer biology by studying in vitro cocultures, in vivo mouse models, and human lung cancer tissue. Lymphocyte conditioned media (CM) induced epithelial-mesenchymal transition (EMT) and migration in both primary human lung cancer cells and cell lines. Correspondingly, major accumulation of Th9 and Th17 cells was detected in human lung cancer tissue and correlated with poor survival. Coculturing lung cancer cells with Th9/Th17 cells or exposing them to the respective CM induced EMT in cancer cells and modulated the expression profile of genes implicated in EMT and metastasis. These features were reproduced by the signatory cytokines IL-9 and IL-17, with gene regulatory profiles evoked by these cytokines partly overlapping and partly complementary. Coinjection of Th9/Th17 cells with tumor cells in WT, Rag1-/-, Il9r-/-, and Il17ra-/- mice altered tumor growth and metastasis. Accordingly, inhibition of IL-9 or IL-17 cytokines by neutralizing antibodies decreased EMT and slowed lung cancer progression and metastasis. In conclusion, Th9 and Th17 lymphocytes induce lung cancer cell EMT, thereby promoting migration and metastatic spreading and offering potentially novel therapeutic strategies.

Abstract B077: Ribosomal RNAs are fragmented into short RNAs in a manner that depends on a person’s sex, population origin, and race: implications for health disparities and personalized medicine

Abstract Ribosomal RNAs (rRNAs) are the most abundant RNAs in eukaryotic cells. rRNAs combine with ribosomal proteins to form the scaffold for the small and large subunits of the ribosomes. There are six distinct rRNAs: four are encoded by the nuclear and two by the mitochondrial genome. Deep sequencing showed that short RNAs, known as “rRNA-derived fragments” or rRFs, arise from all six rRNAs. The accumulated evidence suggests that rRFs have key functional roles. Various studies showed that rRFs can modulate intracellular ATP levels, participate in the response to stress, affect ERK signaling, and are required for DNA repair. Moreover, rRFs can be loaded on Argonaute thereby entering the RNAi pathway and regulating transcript abundance. To systematically study rRFs, we developed a computational pipeline and used it to analyze the short RNA profiles of lymphoblastoid cell lines obtained from 434 healthy participants of the 1000 Genomes Project. The 434 individuals represent five human populations, and two races. Men and women are represented evenly in each of the five populations. We analyzed the rRF profiles for each of the six rRNAs in turn, and separately for the five populations and two sexes. Across the 434 individuals, we identified more than 18,000 rRFs each with a normalized abundance ≥ 10 reads per million. Each of the six rRNAs gives rise to ‘clouds of rRFs’ whose identities and abundances differ characteristically from each rRNA to the next. We found that the clouds of rRFs are produced in a population-specific, race-specific, and sex-specific manner. Like samples, i.e. samples belonging to the same sex and population group, produce rRFs with the same identities and relative abundances, which suggests that rRFs are produced in a regimented manner by currently unknown processes. Preliminary analyses of rRFs in primary cancer samples and cell lines led to similar results. Northern blotting allowed us to independently confirm several of our computational findings. Our laboratory has long focused on the study of microRNAs (miRNAs), miRNA isoforms (isomiRs), transfer RNAs (tRNAs), and tRNA-derived fragments (tRFs) in health and disease. IsomiRs and tRFs play important roles in cells by modulating transcript abundance through Argonaute-loading, translation-stalling, decoying of RNA binding proteins, etc. We were first to show that isomiRs and tRFs are produced constitutively in human tissues and that their profiles depend on a person’s sex, population origin, and race, as well as on tissue type, tissue state, and disease. We also reported sex- and/or race-dependent differences for multiple cancers, including those of the prostate, bladder, lung, kidney, and breast. Through the current study, we show for a third category of short regulatory RNAs, the rRFs, that they too depend on personal attributes such as sex, population origin, and race. The finding and the large number of rRFs have implications for the study of health disparities, in cancer and other diseases, and for efforts to practice personalized medicine. Citation Format: Tess Cherlin, Rogan Magee, Yi Jing, Phillipe Loher, Venetia Pliatsika, Isidore Rigoutsos. Ribosomal RNAs are fragmented into short RNAs in a manner that depends on a person’s sex, population origin, and race: implications for health disparities and personalized medicine [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B077.

A High-Throughput Image Cytometry Method for the Formation, Morphometric, and Viability Analysis of Drug-Treated Mammospheres.

The nonadherent mammosphere assay has been commonly used to investigate cancer stem cell activities in breast cancers that have the ability to form tumorspheres and maintain tumor growth. The sphere formation step is critical, in that it enables the construction of the mammosphere models for downstream assays. The mammosphere assay has also been used to assess the effects of drug treatment on the tumorspheres formed from primary cancer cells or cell lines. Traditionally, the mammosphere formation has been evaluated by standard microscopy systems that required external software for additional analyses. However, this method can be time-consuming and low-throughput, thus impractical for high-throughput characterization of mammosphere models and screening for potential therapeutic cancer drugs. To overcome these challenges, we developed a plate-based high-throughput method to rapidly analyze mammospheres in whole wells using the Celigo Image Cytometer. The method is employed to characterize mammosphere formation and morphology for adherent and nonadherent propagation of four breast cancer cell lines (MCF7, MDA-MB-436, MDA-MB-231, and SKBR3). Next, the dose-dependent effects of four small molecule drugs (doxorubicin, paclitaxel, 8-quinolinol, and salinomycin) are characterized based on sphere formation and viability stained with calcein AM and propidium iodide. We observed growth and morphometric differences between adherent and nonadherent propagation of the four cell lines. Furthermore, drug treatments induced various effects on mammosphere formation, morphology, and viability. The proposed image cytometry method provides a useful tool suitable for high-throughput characterization and analysis of mammospheres, which can improve assay efficiency when investigating the formation capabilities and drug-induced cytotoxicity effects.