Established Cancer Cell Lines Research Articles

Cell State Transitions and Phenotypic Heterogeneity in Luminal Breast Cancer Implicating MicroRNAs as Potential Regulators.

Luminal breast cancer subtypes respond poorly to endocrine and trastuzumab treatments due to cellular heterogeneity arising from the phenotype transitions, accounted for mainly by the loss of receptor expression. The origins of basal-like and human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer subtypes have been attributed to genetic and protein modifications in stem-like cells and luminal progenitor cell populations, respectively. The post-transcriptional regulation of protein expression is known to be influenced by microRNAs (miRNAs) that are deemed to be master regulators of several biological processes in breast tumorigenesis and progression. Our objective was to identify the fractions of luminal breast cancer cells that share stemness potentials and marker profiles and to elucidate the molecular regulatory mechanism that drives transitions between fractions, leading to receptor discordances. Established breast cancer cell lines of all prominent subtypes were screened for the expression of putative cancer stem cell (CSC) markers and drug transporter proteins using a side population (SP) assay. Flow-cytometry-sorted fractions of luminal cancer cells implanted in immunocompromised mice generated a pre-clinical estrogen receptor alpha (ERα+) animal model with multiple tumorigenic fractions displaying differential expression of drug transporters and hormone receptors. Despite an abundance of estrogen receptor 1 (ESR1) gene transcripts, few fractions transitioned to the triple-negative breast cancer (TNBC) phenotype with a visible loss of ER protein expression and a distinct microRNA expression profile that is reportedly enriched in breast CSCs. The translation of this study has the potential to provide novel therapeutic miRNA-based targets to counter the dreaded subtype transitions and the failure of antihormonal therapies in the luminal breast cancer subtype.

Thymic stromal lymphopoietin-stimulated CD4+ T cells induce senescence in advanced breast cancer.

Thymic Stromal Lymphopoietin (TSLP) plays a prominent role in inducing type 2 immune response, commonly associated with atopic diseases. TSLP-activated CD4+ T helper 2 cells block early carcinogenesis by inducing terminal differentiation in spontaneous breast and lung cancer models. However, the impact of TSLP induction on advanced cancer with altered cellular phenotypes is unclear. Using an established MMTV-PyMttg breast cancer cell line, we demonstrate that TSLP-stimulated CD4+ T cells possess an antitumor effect in advanced breast cancer. In contrast to early breast cancer suppression, the antitumor immunity mediated by TSLP-stimulated CD4+ T cells in advanced breast cancer is mediated by the induction of a senescent-like phenotype in cancer cells. Inflammatory CD4+ T cells drive breast cancer cells into senescence by releasing interferon-gamma and tumor necrosis factor-alpha, which directly bind to their receptors on cancer cells. Our findings reveal a novel mechanism of TSLP-activated CD4+ T cell immunity against advanced breast cancer, mediated by cellular senescence as a distinct effector mechanism for cancer immunotherapy.

Tub-010, a Novel Antibody-Drug-Conjugate with Reduced Nonspecific Toxicity Profile Based on Tub-Tag Technology Widens the Therapeutic Window for the Treatment of CD30+ Malignancies

Introduction: Patients with R/R CD30+ malignancies such as PTCL, CTCL and cHL often lack effective and tolerable therapy options. Available treatments frequently provide only short-term disease control at the cost of significant or cumulative toxicities. Brentuximab-Vedotin (BV, Adcetris®), an MMAE-delivering anti-CD30-ADC, has a high response rate but dose-limiting AEs often result in premature dose reduction or even therapy discontinuation. The maleimide chemistry results in a heterogeneous product (DAR 0-8) with varying PK and potency properties and spontaneous deconjugation can cause premature transfer of linker payload to serum proteins and thereby reduce the tumor selectivity. In consequence, some of the BV-induced toxicity is likely design-based leading to CD30-independent cellular uptake and aggregation of the ADC. Objectives: We aimed to generate a novel CD30-ADC with improved biophysical properties to widen the therapeutic window using Tub-tag-conjugation technology. Material & methods: TUB-010 is a next-generation ADC, implementing a novel conjugation strategy, called Tub-tag® conjugation (Schumacher, Angewandte Chemie 2015). The recombinant Tub-tag sequence, a 14 amino acid peptide derived from α-tubulin and fused to the C-termini of the CD30 mAb light chains, has unique features in that it provides a significant degree of hydrophilicity to counterbalance the payload-derived hydrophobicity, thus enabling superior biophysical properties and product homogeneity. This new technology was used to generate a DAR2-ADC with stably linked MMAE via a Cathepsin B cleavable linker (vc-PAB) and chemoenzymatic conjugation of linker payload using Tubulin-tyrosine ligase (TTL). The resulting ADC was tested for physicochemical properties, homogeneity, stability, internalization, preclinical efficacy, nonspecific uptake and general cytotoxicity in primary cells, cell lines, rats & cynomolgus monkeys. Results: Tub-tag technology enabled us to create a DAR2 anti-CD30-ADC with a binding affinity, internalization and lysosomal release characteristics similar to BV. In contrast to BV, TUB-010 is a homogeneous DAR2 ADC. The linker-payload is chemically stably attached to the mAb with neglectable premature deconjugation while in circulation, whereas protease-mediated release of MMAE within the lysosome occurs normally as expected with the vc-PAB linker. The increased ADC stability prior to internalization via CD30 leads to reduced nonspecific transfer of linker-payload to serum proteins, and overall improved PK parameters in rats. The hydrophobicity of MMAE is counterbalanced by hydrophilicity provided by the Tub-tag peptides, which resulted in > 5-fold reduced ADC aggregation under stress conditions compared to BV. The increased hydrophilicity of the overall ADC reduced nonspecific cellular uptake and consecutive cellular toxicity vs BV within the clinically therapeutic range by factor 2- to >10-fold in various human primary cells (Fig. A). When normalized to the MMAE concentration, TUB-010 showed similar in vitro cytotoxic efficacy as well as similar bystander activity compared to BV on established cancer cell lines from cHL, PTCL and CTCL patients with various levels of CD30 expression (Fig. A). In vivo, TUB-010 exerted significantly improved tumor control compared to BV when both ADCs were dosed at equal MMAE concentration in the Karpas-299 cell-derived ALCL xenograft model (Fig. B). In cynomolgus monkey dose range finding and GLP toxicity studies, TUB-010 administered 2 or 4 times Q3W in a dose range of 6-15 mg/kg showed a 5-fold widened therapeutic window for the ADC as the MTD was not reached. Hematological toxicity was reduced compared to BV and no signs of anatomical of functional tissue damage were detectable. Conclusions: Tub-tag technology allows the reduction of nonspecific target-independent toxicity of ADCs in preclinical models by improving key design issues in current ADCs such as stability and overall biophysical features. TUB-010 is a promising, novel and potential best-in-class CD30-ADC which can deliver the cytotoxic payload to CD30+ malignancies with higher precision and with a wider therapeutic window than BV. These features suggest that TUB-010 may increase the clinical benefit with CD30-targeting ADC therapy for patients with CD30+ malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oncolytic Urabe mumps virus: A promising virotherapy for triple-negative breast cancer

Historically, the clinical utility of oncolytic virotherapy as a treatment for a wide range of cancer types was first demonstrated by three pilot human clinical trials conducted in Japan in the 1970s and 1980s using a wild-type Urabe mumps virus (MuV) clinical isolate. Using a sample of the actual original oncolytic Urabe MuV clinical trial virus stock (MuV-U-Japan) used in these Japanese clinical trials, we found that MuV-U-Japan consisted of a wide variety of very closely related Urabe MuVs that differed by an average of only three amino acids. Two MuV-U-Japan isolates, MuV-UA and MuV-UC, potently killed a panel of established human breast cancer cell lines invitro, significantly extended survival of nude mice with human triple-negative breast cancer (TNBC) MDA-MB-231 tumor xenografts invivo, and demonstrated significant killing activity against breast cancer patient-derived xenograft (PDX) cell lines grown as 3D organoids, including PDXs from patients resistant to anthracycline- and taxane-based chemotherapy. We also report success in developing a large-scale MuV-U production and purification process suitable for supporting Investigational New Drug applications for clinical trials. This study demonstrates the suitability of the MuV-UC virus for translation to modern clinical trials for treating patients with TNBC.

Proteome alterations during clonal isolation of established human pancreatic cancer cell lines

Clonal isolation is an integral step of numerous workflows in genome editing and cell engineering. It comprises the isolation of a single progenitor cell from a defined cell line population with subsequent expansion to obtain a monoclonal cell population. This process is associated with transient loss of cell–cell contacts and absence of a multicellular microenvironment. Previous studies have revealed transcriptomic changes upon clonal isolation with cell line specific extent. Since transcriptome alterations are only partially reflected on the proteome level, we sought to investigate the impact of clonal isolation on the cellular proteome to a depth of > 6000 proteins in three established pancreatic cancer cell lines. We show that clonal isolation does have an impact on the cellular proteome, however, with cell line specific extent, affecting different biological processes, and also depending on the isolation method. We demonstrate a different impact of clonal isolation on mesenchymal- and epithelial-derived cell lines mainly affecting cell proliferation, metabolism, cell adhesion and cellular stress. The results bear relevance to the field of genomic editing and cell engineering and highlight the need to consider the impact of clonal isolation when interpreting data stemming from experiments that include this step.

CTPC, a combined transcriptome data set of human prostate cancer cell lines.

Cell lines are the most used model system in cancer research. The transcriptomic data of established prostate cancer (PCa) cell lines help researchers explore differential gene expressions across the various PCa cell lines. Through large scale datamining, we established a curated Combined Transcriptome dataset of PCa Cell lines (CTPC) which contains the transcriptomic data of 1840 samples of 9 commonly used PCa cell lines including LNCaP, LNCaP-95, LNCaP-abl, C4-2, VCaP, 22Rv1, PC3, DU145, and NCI-H660. The CTPC dataset provides an opportunity for researchers to not only compare gene expression across different PCa cell lines but also retrieve the experiment information and associate the differential gene expression data with meta data, such as gene manipulation and drug treatment information. Additionally, based on the CTPC dataset, we built a platform for users to visualize the data (https://pcatools.shinyapps.io/CTPC_V2/). It is our hope that the combined CTPC dataset and the user-friendly platform are of great service to the PCa research community.

EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)

EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)

Abstract 3088: The efficient utilization of paracrine support from established cell lines for breast/ovarian cancer model generation

Abstract Ex vivo cancer cell models provide the starting material for in-depth mechanistic studies of cancer. However, the clinical/histopathologic, biomarker, and genetic heterogeneity of breast cancer has not been well represented in the current breast cancer cell model collection. While published breast cancer model generation protocols have been helpful, their high failure rates indicate the urgent need to improve model derivation efficiency. Here, the Broad Cancer Cell Line Factory (CCLF) project presents a novel model derivation technology to generate breast and ovarian cancer organoids with a high success rate by leveraging the paracrine support from historical cancer cell lines. We observed that most established breast cancer cell lines can grow in a simple basal media with 10% fetal bovine serum; We hypothesized that historical cell lines may secrete vital growth factors that support breast cancer cells' survival and growth. To test this, we randomly selected a pool of 20 breast cancer cell lines, collected its conditioned media (CM20) and incorporated the CM20 as a supplement into our empirical rich media matrix (HYBRID, 16 mixed media conditions) with a Matrigel culturing system. Three-dimensional (3D) structures formed at Day 14-21 in the CM20 supplementary conditions compared to conditions without CM20 and only organoids with the CM20 supplement could be propagated to passage 5 and beyond. We performed pan-cancer targeted sequencing to evaluate tumor content of these organoids at passage 5 with paired tumor tissues. In our first 10 attempts, 95% of organoid cultures were genomically verified as high purity tumor models, indicating the CM20 is essential to enrich breast cancer cell growth in an in vitro culturing setting. We applied the CM20 to ovarian cancers and observed a similar success rate suggesting a tissue-specific supporting manner. We tested conditioned media collected from other historical cancer cell lines but the breast/ovarian cancer organoid growth effect was not recapitulated. Importantly, when testing the individual breast cancer cell lines from the pool of 20, we discovered one cell line to be supporting the effect. More biochemistry work is needed to dissect the possible factors secreted by the line and molecular mechanisms of cancer cell survivors but preliminary data suggests the secretion factors are most likely proteins. We generated 27 breast/ovarian cancer cell models using this technology and RNAseq data shows the breast cancer organoids still express their expected molecular subtype markers. 22 breast/ovarian cancer organoids have been propagated long-term with 17(out of 22) deposited to ATCC. Overall, this method provides an efficient model generation rate for female cancers. We anticipate that this method will not only allow us to quickly increase breast cancer cell model diversity but shed light on a new direction for breast cancer dependencies Citation Format: Rebecca Deasy, Xin Jin, Pierre Michel Jean Beltran, Adel Atari, Madison Liistro, Cheryl Thompson, Stefanie Avril, Julie Boerner, Payal Pradhan, Samuel Klempner, Keith Ligon, William Sellers, Steven Carr, Todd Golub, Yuen-Yi (Moony) Tseng. The efficient utilization of paracrine support from established cell lines for breast/ovarian cancer model generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3088.

Abstract 5684: Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress

Abstract Cancer cells survive under extreme environments that alter protein homeostasis, thereby provoking cellular stress responses, such as the unfolded protein response (UPR) and the integrated stress response (ISR). These pathways increase the expression of molecular chaperones to prevent the accumulation of toxic proteins in the cell. One molecular chaperone in particular, Hsp70, plays a central role in both cellular protection and the response to proteotoxic insults. This is perhaps most evident in cancer cells, some of which overexpress Hsp70 and thrive even when harboring high levels of misfolded, aggregated, and/or unassembled protein complexes. In an effort to define how cells compensate for compromised proteostasis, we examined the adaptive response in breast cancer cells after challenge with a specific Hsp70 inhibitor. We first showed that established breast cancer cell lines bin into classes based on their sensitivity to the drug. We next discovered that resistant cells have higher autophagy levels compared to the more sensitive lines. Moreover, gene and protein expression studies and imaging methods showed that autophagy was further induced by Hsp70 inhibition in resistant breast cancer cells. Consistent with the fact that Hsp70 normally targets aberrant proteins for degradation, our data indicated that resistance to compromised Hsp70 activity arose from elevated levels of autophagy. We next showed that Hsp70 inhibition specifically triggers the ISR pathway. Unexpectedly, protection against the Hsp70 inhibitor was dependent on the ISR sensor, GCN2, which is most commonly associated with the amino acid starvation response. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating the PERK pathway. These data reveal an unexpected route through which cancer cells can adapt to chaperone targeted-proteotoxicity and position GCN2 and autophagy as critical compensatory mechanisms for cancer cells when proteostasis is compromised. Citation Format: Sara Sannino, Jeffrey L. Brodsky. Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5684.

Abstract 1533: Long non-coding RNA control of cancer cell growth

Abstract Long non-coding RNAs (lncRNAs) are involved in controlling regulatory networks critical for gene expression, cellular growth, and development. Altered expression of lncRNAs are associated with tumor progression in multiple types of human cancers, but the mechanisms by which lncRNAs may control growth and proliferation in cancer cells remain unknown in most cases. Development of interventions targeting non-coding RNA regulation of cell growth would open new avenues for cancer treatment. To identify growth regulatory pathways controlled by non-coding RNAs, we used proteomics and genomics tools to study the effect of changes in lncRNA expression levels in human cancer cell lines. First, we identified five growth regulator lncRNAs based on data mining of CRISPR/Cas9 screens and high throughput sequencing studies from patient tumor and normal cells. These lncRNAs have been associated with progression and metastasis in breast, lung and colon cancers. We hypothesized that growth regulator lncRNAs play a role in cancer development by recruiting effector proteins to regulate gene expression. Next, we evaluated the cellular growth phenotype for each lncRNA in overexpression and knockdown strains using multiple established cancer cell lines. We analyzed cellular transcriptome changes after lncRNA perturbation using high-throughput RNA sequencing. Finally, RNA-protein interactions for each growth regulator lncRNA were identified using an RNA hybridization capture method paired with mass spectrometry that enables purification of direct and specific endogenous RNA-binding proteins. Using this combination of transcriptomics and proteomics data, we discovered that perturbation of growth regulator lncRNAs results in dysregulation of growth signaling pathways and increased expression of p53. We are currently identifying the sequence and structure determinants of RNA-protein complex formation to uncover the mechanisms of action of lncRNAs in controlling cancer cell growth. Citation Format: Tong Su, Bobby Kong, Calvin Huang, Jonathan Zhu, Colleen McHugh. Long non-coding RNA control of cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1533.

Abstract 6241: Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment

Abstract Although the established cancer cell lines exhibit less degrees of intratumoral heterogeneity when compared to human solid tumors, studies have suggested that, even within the same cancer cell line, substantial degree of heterogeneity may exist. In this study, we have performed single cell cloning experiment using 4T1 mouse mammary carcinoma cell line and compared the phenotypes of different clones. When compared to the parental 4T1 cell line, one clone (C12) showed markedly different phenotype by exhibiting less aggressive features in vitro and significantly slower tumor growth and metastasis in vivo. The tumor tissue of the C12 showed significant alteration in immune microenvironment as higher number of recruited immune cells including T and B cells. RNA sequencing of the C12 clones and parental 4T1 cells identified Macc1 as a significantly up-regulated gene in parental 4T1 cells. Metastasis-associated colon cancer 1 (MACC1) has been associated with poor prognosis and the promotion of metastasis within several types of cancer, especially colon cancer. The gene encoding the hepatocyte growth factor (HGF) receptor, MET, is a transcriptional target of MACC1. Next, we established Macc1-silenced 4T1 cells using the parental 4T1 cells and observed that Macc1-silencing resulted in a similar phenotype seen in the C12 clone; decreased cell proliferation and migration in vitro and slow tumor growth and metastasis in vivo. To mimic the metastasis in vitro, intravasation was determined as which tumor cells were co-cultured with HUVEC, human umbilical vein endothelial cells. We discovered tumor cell penetration into HUVEC cells was blocked in Macc1-silencing 4T1 group. According to CD45, CD3, and CD19 immunohistochemistry, we also defined immune cells, especially T and B cells, were highly infiltrated in Macc1-silencing tumor like as C12 clone tumor. These results were also reconstructed in EMT6, another TNBC mouse cell line model. In this study, we show that MACC1 can regulate tumor progression and metastasis by remodeling of immune microenvironment. Citation Format: Hye-Youn Son, Woo Hang Heo, Song Bin Li, Mingji Quan, Yu Jeong Her, Kyu-Jin Lee, Ju Hee Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6241.

Starvation mediates pancreatic cancer cell sensitivity to ferroptosis via ERK1/2, JNK and changes in the cell mesenchymal state.

Pancreatic cancer is a highly metastatic and therapy-resistant disease. In the present study, the prospects of a novel approach to kill pancreatic cancer cells were examined: Starvation combined with ferroptosis induction. Established pancreatic cancer cell lines (Miapaca2, Panc-1, Su.86.86 and T3M4), as well as a unique cell line, Capan-26, which was originally derived in the authors' laboratory, were used. Cells were deprived from growth factors, amino acids and pseudo-starved using treatment with mTOR inhibitors; erastin was used to induce ferroptosis. Cell viability and lipid peroxidation measurements using flow cytometry revealed that the starved pancreatic cancer cells reacted differently to ferroptosis induction: The Panc-1, Su.86.86 and T3M4 cells gained sensitivity, while the Miapaca2 cells acquired resistance. Fluorescence microscopy revealed that ERK1/2 translocated to the nucleus of the starved pancreatic cancer cells. Moreover, ERK1/2 pharmacological inhibition with SCH772984 prevented erastin-induced ferroptosis in the starved Panc-1, Su.86.86 and T3M4 cells. Confocal microscopy also indicated JNK activation. However, the inhibition of this kinase revealed its unexpected role in oxidative stress management: Treatment with the JNK inhibitor, SP600125, increased the viability of pseudo-starved cells following erastin treatment. In addition, the FBS-starved Miapaca2 and Capan-26 cells transitioned between epithelial and mesenchymal cell states. The results were further confirmed using wound healing assays, western blot analysis and microscopic analysis of epithelial-to-mesenchymal transition (EMT) markers. Mesenchymal properties were associated with a higher sensitivity to erastin, whereas epithelial-like cells were more resistant. Finally, it was demonstrated that compounds targeting EMT-related signaling pathways increased cell sensitivity to erastin. On the whole, these results confirm that in starved pancreatic cancer cells, ERK1/2 and JNK signaling, as well as switching between epithelial and mesenchymal states mediates sensitivity to erastin and reveal novel therapeutic prospects of the combination of starvation with ferroptosis induction.

Effects of Anticancer Agent P-bi-TAT on Gene Expression Link the Integrin Thyroid Hormone Receptor to Expression of Stemness and Energy Metabolism Genes in Cancer Cells.

Chemically modified forms of tetraiodothyroacetic acid (tetrac), an L-thyroxine derivative, have been shown to exert their anticancer activity at plasma membrane integrin αvβ3 of tumor cells. Via a specific hormone receptor on the integrin, tetrac-based therapeutic agents modulate expression of genes relevant to cancer cell proliferation, survival and energy metabolism. P-bi-TAT, a novel bivalent tetrac-containing synthetic compound has anticancer activity in vitro and in vivo against glioblastoma multiforme (GBM) and other types of human cancers. In the current study, microarray analysis was carried out on a primary culture of human GBM cells exposed to P-bi-TAT (10−6 tetrac equivalent) for 24 h. P-bi-TAT significantly affected expression of a large panel of genes implicated in cancer cell stemness, growth, survival and angiogenesis. Recent interest elsewhere in ATP synthase as a target in GBM cells caused us to focus attention on expression of genes involved in energy metabolism. Significantly downregulated transcripts included multiple energy-metabolism-related genes: electron transport chain genes ATP5A1 (ATP synthase 1), ATP51, ATP5G2, COX6B1 (cytochrome c oxidase subunit 6B1), NDUFA8 (NADH dehydrogenase (ubiquinone) FA8), NDUFV2I and other NDUF genes. The NDUF and ATP genes are also relevant to control of oxidative phosphorylation and transcription. Qualitatively similar actions of P-bi-TAT on expression of subsets of energy-metabolism-linked genes were also detected in established human GBM and pancreatic cancer cell lines. In conclusion, acting at αvβ3 integrin, P-bi-TAT caused downregulation in human cancer cells of expression of a large number of genes involved in electron transport and oxidative phosphorylation. These observations suggest that cell surface thyroid hormone receptors on αvβ3 regulate expression of genes relevant to tumor cell stemness and energy metabolism.

Heat stress stimulates colon cancer cells to secret specific population of extracellular nanovesicles enriched by HSP70 and microRNAs

Background. Heat stress (HS) induces the cellular secretion of heat shock proteins (HSP ) and extracellular nanovesicles (ENVs). The biological link between these phenomena is poorly understood. In the case of colorectal cancer (CRC) cells, the secretion of HSP s and ENV may be involved in the clinical response to intraperitoneal therapy of peritoneal carcinomatosis.Material and Methods. Established colon cancer cell lines COLO 320, HCT 116, HT29 and DLD 1 were used. ENVs were isolated from culture media by differential ultra-centrifugation and analyzed by dynamic light scattering, nanoparticle tracking analysis, atomic force microscopy and flow cytometry. Super-paramagnetic particles (SPMP ) covered by antibodies to the membrane form of Hsp70 were used for isolation and quantification of Hsp70(+) ENVs. Vesicular microRNA was assayed by RT-qPC R.Results. HS induces the secretion of ENVs by CRC cells, the resistance to HS correlates with the activity of HS-induced ENVs secretion. HS induces the secretion of a specific population of ENVs enriched by membrane form Hsp70 (mHsp70). The microRNA content of mHsp70(+) ENVs has qualitative and quantitative features. The concentration of miR-126-3p, -181-5p, -155-5p, -223 is increased in mHSP 70(+) ENVs secreted by three CRC cell lines.Conclusion. HS induces the secretion of mHSP 70(+) ENVs by CRC cells. This phenomenon may be involved in a clinical response to intraperitoneal chemo-hyperthermic perfusion therapy of peritoneal carcinomatosis.

A high-throughput screening assay for identification of chemicals with liver tumor promoting potential using a transgenic zebrafish line

Traditional high-throughput methods for identification of chemicals with liver tumor promotion potentials are based on established cancer cell lines, and rapid and cost-effective high-throughput screening assays in whole organisms are presently lacking. In this study, a transgenic zebrafish liver cancer model was employed to develop a method that could be used to identify chemicals with liver tumor promotion effect quickly and accurately. The method consisted of three parts, including exposure preparation, exposure process and image acquisition. In brief, after chemical exposure for 7 days, 96-well plate exposure system for zebrafish larvae was assessed by microplate reader. Then, the liver cancer promoting potential chemicals were evaluated by field area and field average intensity of fluorescence. The results were further validated by conducting histopathological examination. Our data demonstrated that the high-throughput screening assay developed in this study was reproducible and could be used to rapidly screen chemicals with liver tumor promoting potentials by using tris-(2-chloropropyl)-phosphate (TDCIPP) as a positive control. Furthermore, some other positive chemicals found in previous studies and environmental compounds were assessed using the established method. Results indicated that 86.7% of the positive chemicals and five environmental compounds out of seventeen compounds could enhance liver tumor progression.

Type-2 cGMP-dependent protein kinase suppresses proliferation and carcinogenesis in the colon epithelium.

A large body of evidence has demonstrated that cyclic-guanosine monophosphate (cGMP), signaling has anti-tumor effects that might be used for colon cancer prevention. The tumor-suppressive mechanism and the signaling components downstream of cGMP remain largely unknown. The present study has characterized the expression of cGMP-dependent protein kinases (PKG1, PKG2) in normal and cancerous tissue from human colon. PKG1 was detected in both normal and tumor tissue, where it localized exclusively to the lamina propria and stroma (respectively). In contrast, PKG2 localized specifically to the epithelium where its expression decreased markedly in tumors compared to matched normal tissue. Neither PKG isoform was detected at the RNA or protein level in established colon cancer cell lines. To test for a potential tumor-suppressor role of PKG2 in the colon epithelium, Prkg2 knockout (KO) mice were subjected to azoxymethane/dextran sulfate-sodium (AOM/DSS) treatment. PKG2 deficiency was associated with crypt hyperplasia (Ki67) and almost twice the number of polyps per mouse as wild-type (WT) siblings. In vitro culture of mouse colon epithelium as organoids confirmed that PKG2 was the only isoform expressed, and it was detected in both proliferating and differentiating epithelial compartments. Colon organoids derived from Prkg2 KO mice proliferated more rapidly and exhibited a reduced ability to differentiate compared to WT controls. Taken together our results highlight PKG2 as the central target of cGMP in the colon, where it suppresses carcinogenesis by controlling proliferation in an epithelial-cell intrinsic manner.

The 3D in vivo chorioallantoic membrane model and its role in breast cancer research.

We aimed to evaluate the role of the chorioallantoic membrane model (CAM) in breast cancer research. The following is an overview of the use of the CAM in the field of breast cancer research based on a PubMed literature query. The CAM is a 3D in vivo model that can be used for the analysis of tumor growth, biology and angiogenesis of primary tumor tissue or tumor cell lines. The CAM model has been used in breast cancer research for drug testing, migration assays and the evaluation of vascularization, amongst others. The CAM model is a valuable method that offers a better imitation of the physiological phenomena compared to 2D or 3D in vitro models. The CAM model has primarily and successfully been utilized for the assessment of the tumor biology of established breast cancer cell lines. Further, the CAM model is a promising method to analyze patient derived primary tumor material and could be used as a "patient-specific 3D-tumor-therapy-model" for the cost-efficient evaluation of anti-cancer drugs to find the optimal treatment for breast cancer patients.

Selenium Induces Pancreatic Cancer Cell Death Alone and in Combination with Gemcitabine.

Survival rate for pancreatic cancer remains poor and newer treatments are urgently required. Selenium, an essential trace element, offers protection against several cancer types and has not been explored much against pancreatic cancer specifically in combination with known chemotherapeutic agents. The present study was designed to investigate selenium and Gemcitabine at varying doses alone and in combination in established pancreatic cancer cell lines growing in 2D as well as 3D platforms. Comparison of multi-dimensional synergy of combinations’ (MuSyc) model and highest single agent (HSA) model provided quantitative insights into how much better the combination performed than either compound tested alone in a 2D versus 3D growth of pancreatic cancer cell lines. The outcomes of the study further showed promise in combining selenium and Gemcitabine when evaluated for apoptosis, proliferation, and ENT1 protein expression, specifically in BxPC-3 pancreatic cancer cells in vitro.

The Mitochondrial Disruptor Devimistat (CPI-613) Synergizes with Genotoxic Anticancer Drugs in Colorectal Cancer Therapy in a Bim-Dependent Manner.

Colorectal cancer is one of the most frequent tumor entities, with an increasing incidence and mortality in younger adults in Europe and the United States. Five-year survival rates for advanced colorectal cancer are still low, highlighting the need for novel targets in colorectal cancer therapy. Here, we investigated the therapeutic potential of the compound devimistat (CPI-613) that targets altered mitochondrial cancer cell metabolism and its synergism with the antineoplastic drugs 5-fluorouracil (5-FU) and irinotecan (IT) in colorectal cancer. Devimistat exerted a comparable cytotoxicity in a panel of established colorectal cancer cell lines and patient-derived short-term cultures independent of their genetic and epigenetic status, whereas human colonic epithelial cells were more resistant, indicating tumor selectivity. These findings were corroborated in intestinal organoid and tumoroid models. Mechanistically, devimistat disrupted mitochondrial membrane potential and severely impaired mitochondrial respiration, resulting in colorectal cancer cell death induction independent of p53. Combination treatment of devimistat with 5-FU or IT demonstrated synergistic cell killing in colorectal cancer cells as shown by Combenefit modeling and Chou-Talalay analysis. Increased cell death induction was revealed as a major mechanism involving downregulation of antiapoptotic genes and accumulation of proapoptotic Bim, which was confirmed by its genetic knockdown. In human colorectal cancer xenograft mouse models, devimistat showed antitumor activity and synergized with IT, resulting in prolonged survival and enhanced therapeutic efficacy. In human tumor xenografts, devimistat prevented IT-triggered p53 stabilization and caused synergistic Bim induction. Taken together, our study revealed devimistat as a promising candidate in colorectal cancer therapy by synergizing with established antineoplastic drugs in vitro and in vivo.

Abstract PO-227: Role of IL-1β pathway in colon cancer progression and its therapeutic implications in African American colon cancer cell lines

Abstract Background: Colorectal cancer (CRC) is the third most common cancer and third cause of cancer-related death among African Americans (AA) in the US. When compared to Caucasian Americans (CA), AA patients present with higher incidence and mortality rates for CRC. Recent findings indicate that they have reduced response to the standard of care chemotherapeutic agent 5-Fluorouracil (5-FU) as well as lower frequency of MSI tumors, making them also less likely to respond to conventional immunotherapies. Previous results from human genomic analyses suggest that certain differences seen in AA patients might be due to a decreased antitumor immune response as well as an increased expression of genes involved in inflammatory processes, such as Interleukin-1β (IL-1β). Therefore, we aimed to investigate the role of IL-1β in promoting cell proliferation, cell migration, 5-FU resistance and activation of specific inflammatory pathways in novel AA colon cancer cell lines, and how these responses would compare to well established CA colon cancer cell lines. Methods: Our approach includes using MTS colorimetric assay and Transwell assay to examine the effects of IL-1β treatment on cell proliferation and migration. We performed Western Blot analysis to detect expression of phosphoproteins following treatment with IL-1β. We also investigated how IL-1β affects 5-FU resistance in AA and CA colon cancer cell lines in terms of cell viability. Finally, we tested the ability of IL-1 Receptor antagonist (IL-1Ra) to inhibit the effects of IL-1β on cell proliferation, protein expression, and 5-FU response. Results: Our MTS assay results indicated that cell proliferation in response to IL-1β differs between the AA and the CA colon cancer cell lines, with the AA colon cancer cells being more responsive. Whereas, the migration rate is increased after stimulation with IL-1β for both AA and CA colon cancer cell lines. Protein expression of Phospho-IkB-alpha is increased in AA but not CA colon cancer cell lines following IL-1β treatment, while Phospho-JNK expression was different between the cell lines following treatment. Importantly, our results show that 5-FU efficacy is decreased in the presence of IL-1β for both AA and CA colon cancer cell lines and that treating the cells with IL-1Ra appeared to reestablish 5-FU cell killing effect. IL-1Ra also suppresses the effects of IL-1β on cell proliferation and protein expression. Conclusions: Taken together, our results demonstrated a differential response to IL-1β for the AA colon cancer cell lines, suggesting a probable role played by the cytokine in driving inflammation-related cancer progression and reveals a possible new target to exploit in immunotherapy for this population. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Jovanny Zabaleta, Jennie Williams, Laura Martello-Rooney. Role of IL-1β pathway in colon cancer progression and its therapeutic implications in African American colon cancer cell lines [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-227.