Clonal Cell Lines Research Articles

Abstract PR007: Identifying targeted therapies for muscle invasive bladder cancer via STAG2 expression

Abstract High stromal antigen 2 (STAG2) protein expression is significantly associated with shorter overall and progression free survival of muscle invasive bladder cancer (MIBC) patients. Unfortunately, STAG2 itself is difficult to target as it functions as a transcriptional regulator and has no intrinsic enzymatic activity. Therefore, it becomes essential to identify downstream therapeutic vulnerabilities based on STAG2 expression. In this study, we accomplish this by employing a drug screen in isogenic STAG2 wild-type (WT) and knock-out (KO) MIBC cell lines, analyzing drug sensitivity of DepMap bladder cancer cell lines, and performing in vitro dose-response experiments based on our drug screen findings. To delineate changes in drug sensitivity due specifically to altered STAG2 expression, we used CRISPR-Cas9 to knock out STAG2 expression in T24 MIBC cells and generated two clonal STAG2 KO cell lines to compare to control. Then, we performed a drug screen with 312 drugs targeting oncogenic signaling pathways. We treated cells with drugs at 534 nM and read viability after 72 hours. To expand our drug screen findings to a larger group of cell lines and drugs, we further queried the DepMap database which includes drug sensitivity data for 4,868 drugs and approximately 2,000 cancer cell lines. We selected drug screened bladder cancer cell lines from DepMap and grouped them into ‘STAG2-high’ or ‘STAG2-low’ cohorts based on STAG2 mRNA expression. We then compared these two groups to identify if either group was more sensitive to drugs in the DepMap database. From these drug screen and DepMap analyses, we identified TAK-733 (MEKi), Berzosertib (ATRi), Prexasertib (CHKi), PI-103 (PI3Ki), and Rigosertib (PLKi) as candidate drugs that are differentially effective based on STAG2 expression level. We performed validation studies with these candidate drugs by treating matched STAG2 WT and KO T24 cells with each drug at concentrations ranging from 0.001 to 100 µM, then computationally modeled EC50 values in R. Cells with STAG2 KO were more sensitive to Berzosertib (T24 Control EC50: 1.78 µM, T24 KO EC50s: 1.03, 0.77 µM) compared to cells with WT STAG2 expression. Interestingly, cells with WT STAG2 expression were more sensitive to PI-103 (T24 Control EC50: 0.33 µM, T24 KO EC50s: 0.97, 1.41 µM) compared to cells with STAG2 KO. This suggests that STAG2 expressing cells may have a unique dependency on PI3K signaling, which has previously been reported in sarcoma cell lines. These findings lay the foundation for further investigation of Berzosertib as a targeted therapy for MIBC patients with mutated or low STAG2, and PI-103 for MIBC patients with high STAG2 who currently suffer worse clinical outcomes. Citation Format: Sarah R. Athans, Henry Withers, Zara Kazmierczak, Katerina Gurova, Anna Woloszynska. Identifying targeted therapies for muscle invasive bladder cancer via STAG2 expression [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR007.

Differential response of injured and healthy retinas to syngeneic and allogeneic transplantation of a clonal cell line of immortalized olfactory ensheathing glia: a double-edged sword.

JOURNAL/nrgr/04.03/01300535-202508000-00029/figure1/v/2024-09-30T120553Z/r/image-tiff Olfactory ensheathing glia promote axonal regeneration in the mammalian central nervous system, including retinal ganglion cell axonal growth through the injured optic nerve. Still, it is unknown whether olfactory ensheathing glia also have neuroprotective properties. Olfactory ensheathing glia express brain-derived neurotrophic factor, one of the best neuroprotectants for axotomized retinal ganglion cells. Therefore, we aimed to investigate the neuroprotective capacity of olfactory ensheating glia after optic nerve crush. Olfactory ensheathing glia cells from an established rat immortalized clonal cell line, TEG3, were intravitreally injected in intact and axotomized retinas in syngeneic and allogeneic mode with or without microglial inhibition or immunosuppressive treatments. Anatomical and gene expression analyses were performed. Olfactory bulb-derived primary olfactory ensheathing glia and TEG3 express major histocompatibility complex class II molecules. Allogeneically and syngenically transplanted TEG3 cells survived in the vitreous for up to 21 days, forming an epimembrane. In axotomized retinas, only the allogeneic TEG3 transplant rescued retinal ganglion cells at 7 days but not at 21 days. In these retinas, microglial anatomical activation was higher than after optic nerve crush alone. In intact retinas, both transplants activated microglial cells and caused retinal ganglion cell death at 21 days, a loss that was higher after allotransplantation, triggered by pyroptosis and partially rescued by microglial inhibition or immunosuppression. However, neuroprotection of axotomized retinal ganglion cells did not improve with these treatments. The different neuroprotective properties, different toxic effects, and different responses to microglial inhibitory treatments of olfactory ensheathing glia in the retina depending on the type of transplant highlight the importance of thorough preclinical studies to explore these variables.

Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.

Clonal cell lines harboring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 6-8 weeks.

Creating new in vitro models to examine the role of Pkd1 in cell and tubular physiology

Autosomal dominant polycystic kidney disease (ADPKD) results from loss of function mutations in polycystin proteins PC1 and PC2 encoded by the PKD1 and PKD2 genes. ADPKD affects 1 in 400 to 1 in 1000 people worldwide, with approximately 50% of patients progressing to end stage renal disease. The physiological function of the polycystins and the mechanisms of cystogenesis remain largely unknown. In vitro models may provide the clearest possibility to understand the immediate cell biological effects of polycystin loss, without the confounding second order injury that occurs as cysts develop. Some of the hurdles to in vitro models include clonal variation, renal segment heterogeneity, and inconsistent growth in 3D culture conditions. To address this need, we isolated renal epithelial cells from either the cortex or the medulla of Pkd1flox/flox; Pax8-rtTA; tetO-7-Cre mice crossed with the immorto-mouse expressing the temperature sensitive SV40 large T antigen. We then cloned single cells into isogenic cell lines, one cortical and one medullary. We verified the induction of Pkd1 KO by treating the cells with either 10 ug/ml doxycycline to knock out (KO) Pkd1 or vehicle control. Treatment continued for 7 days, after which Pkd1 KO was confirmed by western blot. Next, we sought to functionally characterize the two new cell lines. When the cells were grown on transwell supports, cells formed epithelial tight junctions with a trans-epithelial resistance of 2467 ohms/cm2. Resistance increased to 3130 ohms/cm2 following Pkd1 KO in the medullary clone. We next measured the rate of media acidification with or without Pkd1 KO as a proxy for glucose utilization. Pkd1 KO cells acidified faster than controls in both clones. Next, we addressed their ability to grow into tubuloids or spheroids / cysts in 3D culture using multiple approaches. Cells were either 1) suspended directly in Matrigel and plated in a 96 well plate, 2) suspended in Matrigel droplets, with multiple droplets adhering to an individual well of a 6 well plate, 3) plated within a Matrigel “sandwich”, or 4) plated within a sandwich and pulsed with glial derived neurotropic factor (GDNF) for 48 hours. All clones suspended directly in Matrigel formed spheroids, with more and larger spheroids in the Matrigel droplet cultures. Similar results were observed in the cortical clone grown in the sandwich, however the Pkd1 KO medullary clone in the same culture conditions had several giant spheroid structures at least 3 times larger than any other organoid observed in control medullary cultures. Finally, cortical cultures grown in the sandwich and pulsed with GDNF revealed larger spheroid structures in the Pkd1 KO cells than the control structures. Medullary cultures grown with GDNF revealed complicated structures with lumens and tubuloid projections in the controls, and large hollow spheroid / cystic structures in the Pkd1 KO cultures. Taken together, these data support the Matrigel sandwich with GDNF pulse may be the most useful to interrogate physiological changes in the absence of Pkd1 expression. In summary, we have created two new clonal cell lines with inducible Pkd1 KO, with isogenic controls, and the ability to differentiate into complicated tubuloids or spheroids in a genotypical manner, that may help elucidate the most proximate events occuring in renal epithelial cells after the inactivation of Pkd1. U54 DK126114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2050: Slc43a3 And Intracellular Fatty Acid Trafficking

Background: Fatty acids (FAs) have important roles in many cellular functions. Dysregulation of FA flux is both a characteristic and effector of many prevalent clinical conditions, including diabetes mellitus, obesity as well as insulin resistance and/or metabolic syndrome. Adipocytes take up long chain fatty acids through diffusion and protein mediated transport, whereas fatty acid efflux is considered to occur by diffusion. Our previous study suggests the possibility of a protein facilitated/regulated process of FA efflux by demonstrating that the organic anion transport inhibitor DIDS strongly inhibits FA efflux in lipolytically-stimulated adipocytes, leading to an accumulation of intracellular FAs, while not affecting glycerol efflux. We hypothesize that there is potential regulator(s) for FA efflux. Methods and Results: In a screen of subcutaneous adipose samples from obese patients for expression levels of transporters without known function, Slc43a3 (a member of the solute carrier transporter family) shown increased expression after bariatric surgery. In addition, Slc43a3 regulates fatty acid flux in adipocytes. To study the mechanism of Slc43a3 function, clonal lines of preadipocyte OP9 cells were generated, in which green fluorescent protein has been knocked-in at the C-terminus of endogenous Slc43a3 using CRISPR/Cas9. In parallel, Slc43a3 knockout (KO) line has also been generated. Examination of the cells showed that expression of Slc43a3 induced in the cytoplasm of the adipocytes during differentiation. Slc43a3KO adipocytes accumulate bigger lipid droplets and less small lipid droplets. To study the functional significance of Slc43a3 in vivo, adipose tissue specific KO of Slc43a3 were generated with floxed Slc43a3 and tamoxifen induced adiponectin-Cre recombinase. The Slc43a3 KO mice have less body weight and less % body fat on a normal chow diet; however, no differences are noted when the mice are switched to high fat diet, indicating the lack of lipid signaling molecules in the Slc43a3 KO mice with normal chow diet. Functional analysis shows a blunted stimulated lipolysis in the Slc43a3 KO mice. Conclusions: Both in vitro and in vivo data confirmed an unique role of Slc43a3 in regulating fatty acid flux in adipose tissue.

Deletion of PTP4A3 phosphatase in high grade serous ovarian cancer cells decreases tumorigenicity and produces marked changes in intracellular signaling pathways and cytokine release.

The oncogenic protein tyrosine phosphatase PTP4A3 is frequently overexpressed in human ovarian cancers and is associated with poor patient prognosis. PTP4A3 is thought to regulate multiple oncogenic signaling pathways, including STAT3, SRC, and ERK. The objective of this study was to generate ovarian cancer cells with genetically depleted PTP4A3; to assess their tumorigenicity; to examine their cellular phenotype; and to uncover changes in their intracellular signaling pathways and cytokine release profiles. Genetic deletion of PTP4A3 using CRISPR/Cas9 enabled the generation of individual clones derived from single cells isolated from the polyclonal knockout population. We observed a >90% depletion of PTP4A3 protein levels by Western blotting in the clonal cell lines compared to the sham transfected wildtype population. The wildtype and polyclonal knockout cell lines shared similar monolayer growth rates, while the isolated clonal populations 2B4, 3C9, and 3C12 exhibited significantly lower monolayer growth characteristics consistent with their lower PTP4A3 levels. The clonal PTP4A3 knockout cell lines also had substantially lower in vitro colony formation efficiencies compared to the wildtype cells and were less tumorigenic in vivo The clonal knockout cells were markedly less responsive to IL-6-stimulated migration in a scratch wound assay compared to the wildtype cells. Antibody microarray assays documented differences in cytokine release and intracellular phosphorylation patterns in the PTP4A3 deleted clones. Bioinformatic network analyses indicated alterations in cellular signaling nodes. These biochemical changes could ultimately form the foundation for pharmacodynamic endpoints useful for emerging anti-PTP4A3 therapeutics. Significance Statement Clones of high grade serous ovarian cancer cells were isolated in which the oncogenic phosphatase PTP4A3 was deleted using CRISPR/Cas9 methodologies. The PTP4A3 null cells exhibited loss of in vitro proliferation, colony formation, and migration, and reduced in vivo tumorigenesis. Marked differences in intracellular protein phosphorylation and cytokine release were seen. The newly developed PTP4A3 knockout cells should provide useful tools to probe the role of PTP4A3 phosphatase in ovarian cancer cell survival, tumorigenicity and cell signaling.

Abstract LB334: Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis

Abstract Introduction: Eicosanoid mediated mechanisms of hormone therapy resistance in ER+HER2- breast cancer are poorly understood. Cytochrome P450 arachidonic acid (AA) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to breast cancer progression by promoting mitochondrial oxidative phosphorylation (OXPHOS). However, it remains unclear how EETs contribute to hormonal therapy resistance. In this study, the well-known letrozole resistant (LR) MCF-7 AC1 cell line overexpressing aromatase was selected for fulvestrant resistance (FR). A clonal cell line resistant to letrozole and fulvestrant (LR/FR) also exhibited resistance to palbociclib (PR) (MCF-7 AC1 LR/FR/PR). Xenograft tumors of this cell line were resistant to letrozole, fulvestrant, and palbociclib. Structure activity studies and modeling led to the development of CYP3A4 AA epoxygenase inhibiting biguanides hexyl cuban-1-yl biguanide (HCB) and fluorinated hexyl derivatives C5F2-HCB and C6F3-HCB. Fluorination on the hexyl moiety led to broader inhibition of EET regioisomer biosynthesis. These agents were tested for inhibition of proliferation, signaling, and OXPHOS/glycolysis balance in the MCF-7 AC1 LR/FR/PR cell line. Results: In the MCF-7 AC1 LR/FR/PR clonal cell line cyclin D1 and estrogen receptor (ER) expression were undetectable, while cyclin E1, CDK4, CDK6, CYP3A4, and c-MYC were upregulated 11, 40, 13, 10, and 12- fold (n=3, p<0.001) compared to the MCF-7 cell line. LC-MS analysis demonstrated 1.8, 1.4, and 1.2- fold higher total cellular levels of (±)8,9, (±)11,12, and (±)14,15-EET in the MCF-7 AC1 LR/FR/PR cell line compared to the MCF-7 cell line (n=3, p<0.05). C6F3-HCB suppressed cellular levels of EETs: (±)8,9-EET by 55% (n=3, p<0.031), (±)11,12-EET by 53% (n=3, p<0.029), and (±)14,15-EET by 60% (n=3, P<0.0001). The MCF-7 AC1 LR/FR/PR cell line was more potently inhibited by hexyl-cuban-1-yl biguanides compared to the MCF-7 cell line, exhibiting lower IC50 values (MCF-7 AC1 LR/FR/PR IC50 vs. MCF-7 IC50; HCB 4.1 vs. 7.0 uM; for C5F2-HCB 10 vs. 25 uM; C6F3-HCB: 5.4 vs. 12 uM; P values all <0.05). Measurement of spare respiratory capacity revealed that the MCF-7 AC1 LR/FR/PR cell line had 4.2% spare respiratory capacity compared to 34% for MCF-7, indicating a lower OXPHOS reserve for the resistant cells. Additionally, the MCF-7 AC1 LR/FR/PR cell line displayed a 2.4-fold higher extracellular acidification rate (ECAR) than the MCF-7 cell line, indicating a higher glycolysis rate (Warburg effect) in the resistant cells. Conclusion: Selection for fulvestrant resistance of MCF-7 AC1 cells resulted in upregulation of CYP3A4 and biosynthesis of hormone therapy resistance associated EETs while correlating with greater sensitivity to fluorinated hexyl-cuban-1-yl biguanides that inhibit EET biosynthesis. Citation Format: Zhijun Guo, Jianxun Lei, Allison Makovec, Swaathi Jayaraman, John R. Hawse, Carol Lange, Elizabeth Ambrose, Gunda I. Georg, Tony D'Assoro, Goetz P. Matthew, David A. Potter. Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB334.

A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Molecularly generated rat hepatitis E virus strains from human and rat show efficient replication in a human hepatoma cell line

The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Whereas HEV genotypes 1–4 of species Paslahepevirus balayani are commonly found in humans, infections with ratHEV (species Rocahepevirus ratti) were previously considered to be restricted to rats. However, several cases of human ratHEV infections have been described recently. To investigate the zoonotic potential of this virus, a genomic clone was constructed here based on sequence data of ratHEV strain pt2, originally identified in a human patient with acute hepatitis from Hongkong. For comparison, genomic clones of ratHEV strain R63 from a rat and of HEV genotype 3 strain 47832mc from a human patient were used. After transfection of in vitro-transcribed RNA from the genomic clones into the human hepatoma cell line HuH-7-Lunet BLR, virus replication was shown for all strains by increasing genome copy numbers in cell culture supernatants. These cells developed persistent virus infections, and virus particles in the culture supernatant as well as viral antigen within the cells were demonstrated. All three generated virus strains successfully infected fresh HuH-7-Lunet BLR cells. In contrast, the human hepatoma cell lines HuH-7 and PLC/PRF/5 could only be infected with the genotype 3 strain and to a lesser extent with ratHEV strain R63. Infection of the rat-derived hepatoma cell lines clone 9, MH1C1 and H-4-II-E did not result in efficient virus replication for either strain. The results indicate that ratHEV strains from rats and humans can infect human hepatoma cells. The replication efficiency is strongly dependent on the cell line and virus strain. The investigated rat hepatoma cell lines could not be infected and other rat-derived cells should be tested in future to identify permissive cell lines from rats. The developed genomic clone can represent a useful tool for future research investigating pathogenicity and zoonotic potential of ratHEV.

Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβ in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.

DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

X Ray-Induced Insulinoma Cell Line Rin-5F Has a Novel Mutation Site, C.A1459G (P.T487A), in Death Domain Associated Protein (DAXX) Gene

A popular toxicological and pharmacological research cell line is the insulin-secreting pancreatic cell line Rin-5F. The cell line originates from insulinomas induced by X-ray exposure. The author of this report looked at the mutation status of the DAXX gene in the Rin-5F cell line clone.<em> </em>The complete DNA and RNA were extracted from the cultivated cells as well. Double-stranded cDNA was then synthesized using the RNA template. Sequencing was done using a 3730xl DNA Analyzer.<em> </em>In the present study, c.A1459G (p.T487A) in Exon 5 in the DAXX gene was detected in Rin-5F cell lines, one of the X-ray-induced insulinomas. An NCBI homology search reveals that the 487 amino acid site in rats is the 497 amino acid of humans, based on the genomic cDNA homology between rats and humans. In humans, the COSMIC database suggests that mutations involving 497 amino acids have not been detected in all human cancers. However, the mutation of 496 amino acids was detected in human stomach and colon cancers. This is the first account of the DAXX gene's state in a cell line created by exposure to X-rays. This may point to the need for additional data and research on unique gene alterations involved in the development of X-ray-induced insulinoma tumors.

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

Latent macrophage and immature B cell lines generated with hygromycin-resistant murine gammaherpesvirus 68 genome expresses modest levels of viral miRNAs

Murine gammaherpesvirus 68 (MHV68) establishes latency mainly in B cells and causes lymphomas reminiscent of human gammaherpesvirus diseases in laboratory mice. To study the molecular mechanism of virus infection and how the viral determinants control cell and eventually cause tumorigenesis, readily available latently infected cell lines are essential. For in vitro MHV68 latency studies, only two cell culture systems have been available. Gammaherpesviruses are known to infect developing B cells and macrophages, therefore we aimed to expand the MHV68 latently infected cell line repertoire. Here, several latently infected immature B cell and macrophage-like cell line clones were generated. Hygromycin-resistant recombinant MHV68 was isolated from a laboratory-made latent cell line, HE2.1, and propagated to develop stable cell lines that carry the viral genome under hygromycin selection. Subclones of these cells lines were analyzed for viral miRNA expression by TaqMan qPCR and assessed for expression of a lytic viral transcript M3. The cell lines maintain the viral genome as an episome shown by the digestion-circularization PCR assay. Latently infected cell lines generated here do not express viral miRNAs higher than the parental cell line. However, these cell lines may provide an alternative tool to study latency mechanisms and miRNA target identification studies.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

Abstract C016: Pre-existing T cell inflammation is a determinant of response to mesothelin chimeric antigen receptor T cell therapy in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a 12.5% 5-year survival rate and is predicted to become the 2nd leading cause of cancer-related death by 2030. PDAC is known to be resistant to immunotherapy. Thus, while chimeric antigen receptor (CAR) T cell therapy shows the potential to mediate durable responses against some hematologic and solid tumors, in PDAC, the efficacy of CAR T therapy has remained limited. PDAC tumors show heterogenous T cell infiltration with some tumors containing a high number of T cells. As T cell infiltration associates with prolonged survival, we hypothesized that the density of pre-existing T cell infiltration would predict response to CAR T therapy. Methods: Previously established clonal mouse PDAC cell lines, which show a T cell inflamed or non-inflamed tumor microenvironment upon in vivo implantation, were used. Mouse CAR T cells specific to mesothelin, a tumor antigen expressed by PDAC, were generated using retroviral transduction. Mice were implanted subcutaneously with T cell inflamed or non-inflamed tumor cell lines. Ten days later mesothelin CAR T cells were administered intravenously. Flow cytometry and multiplex immunohistochemistry was used to define the density and spatial location of CAR T cell infiltration. The impact of CAR T cells on tumor growth and survival was also measured. Results: CAR T cells were found to traffic to T cell inflamed tumors, which led to delayed tumor outgrowth. However, such results were not seen in T cell non-inflamed tumors. Rather in mice with T cell non-inflamed tumors, CAR T cells accumulated in the liver. Further, CAR T cells effectively eliminated T cell inflamed and non-inflamed tumor cells in vitro, demonstrating that tumor intrinsic mechanisms of resistance to T cell killing were not a major determinant of treatment efficacy. Conclusions: Our findings suggest the therapeutic potential of CAR T cells in PDAC but highlight mechanisms of T cell exclusion as a therapeutic barrier wherein CAR T cells accumulate in host organs. Citation Format: Jacqueline B. Plesset, Heather Coho, Kelly Markowitz, Dhruv Patel, Max M. Wattenberg, Meredith L. Stone, Devora Delman, Gregory L. Beatty. Pre-existing T cell inflammation is a determinant of response to mesothelin chimeric antigen receptor T cell therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C016.

HucMSC-Ex alleviates DSS-induced colitis in mice by decreasing mast cell activation via the IL-33/ST2 axis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease that poses challenges in terms of treatment. The precise mechanism underlying the role of human umbilical cord mesenchymal stem cell-derived exosome (HucMSC-Ex) in the inflammatory repair process of IBD remains elusive. Mucosal mast cells accumulate within the intestinal tract and exert regulatory functions in IBD, thus presenting a novel target for addressing this intestinal disease. A mouse model of Dextran Sulfate Sodium (DSS)-induced colitis was established and hucMSC-Ex were administered to investigate their impact on the regulation of intestinal mast cells. An in vitro co-culture model using the human clonal colorectal adenocarcinoma cell line (Caco-2) and human mast cell line (LAD2) was also established for further exploration of the effect of hucMSC-Ex. We observed the accumulation of mast cells in the intestines of patients with IBD as well as mice. In colitis mice, there was an upregulation of mast cell-related tryptase, interleukin-33 (IL-33), and suppression of tumorigenicity 2 receptor (ST2 or IL1RL1), and the function of the intestinal mucosal barrier related to intestinal tight junction protein was weakened. HucMSC-Ex treatment significantly reduced mast cell infiltration and intestinal damage. In the co-culture model, a substantial number of mast cells interact with the epithelial barrier, triggering activation of the IL-33/IL1RL1 (ST2) pathway and subsequent release of inflammatory factors and trypsin. This disruption leads to aberrant expression of tight junction proteins, which can be alleviated by supplementation with hucMSC-Ex. Our results suggest that hucMSC-Ex may reduce the release of mast cell mediators via the IL-33/IL1RL1 (ST2) axis, thereby mitigating its detrimental effects on intestinal barrier function.

Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

Recombinant CHO Cell Pool Generation Using PiggyBac Transposon System.

CHO cell pools with desirable characteristics of high titer and consistent product quality are useful for rapid production of recombinant proteins. Here, we describe the generation of CHO cell pools using the piggyBac transposon system for mediating gene integration. The method describes the co-transfection of cells with the donor plasmid (coding for the gene of interest) and the helper plasmid (coding for the transposase) using polyethyleneimine (PEI). This is followed by a genetic selection for the generation of a cell pool. The resulting cell pool can be used to start a batch or fed-batch culture. Alternatively, it can be used for generation of clonal cell lines or generation of cell banks for future use.

Whole-Genome CRISPR-Cas9 Screen Identifies ZBTB7A As a Potential Therapeutic Target for Cda-II

Congenital dyserythropoietic anemia type II (CDA-II) is an autosomal recessive disease characterized by anemia, ineffective erythropoiesis, and increased bone marrow bi-nucleated erythroblasts. CDA-II is caused by loss-of-function mutations in SEC23B, which encodes a component of coat protein complex II (COP-II) vesicles/tubules that transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Mammals express two SEC23 paralogs, SEC23A and SEC23B. We have previously shown that SEC23A is functionally interchangeable with SEC23B and that increased expression of SEC23A rescues the SEC23B-deficient erythroid differentiation defect observed in CDA-II. Here, we utilized our recently generated clonal SEC23B deficient HUDEP-2 cell line to identify novel therapeutic targets for CDAII. SEC23B deficient HUDEP2 cells survive and grow normally when cultured as pro-erythroblasts in ‘maintenance media’. However, upon culturing these cells in ‘differentiation media’, which results in semi-synchronous erythroid differentiation, differentiated SEC23B-null HUDEP2 cells exhibit reduced growth, impaired differentiation, increased bi-nucleated erythroid cells, and erythroid cell death, features of CDA-II. To identify novel genes that play important roles in the pathophysiology of CDAII, we developed a functional genome-scale CRISPR knockout screen to define genes that when deleted, rescue the SEC23B-null erythroid differentiation defect. SEC23B null HUDEP2 cells cultured in maintenance media were infected with the hGeCKO-v2 lentiviral library at a multiplicity of infection (MOI) of ~0.3, allowing most transduced cells to receive 1 sgRNA (and Cas9) to knockout 1 gene only. Transduced cells were puromycin selected and passaged in expansion media for 14 days. SEC23B-null HUDEP2 cells were then differentiated for 10 days. Cells were harvested prior to differentiation (Day 0, D0), and differentiated (orthochromatic) cells were sorted at D10 of differentiation. Integrated sgRNAs were quantified by deep sequencing. sgRNAs targeting ZBTB7A were amongst the most enriched in D10 compared to D0 erythroid cells. To validate these findings, we transduced SEC23B-null HUDEP2 cells with two independent and efficient ZBTB7A-targeting sgRNAs. We found that ZBTB7A deletion (using either of the 2 sgRNAs) rescued the lethality and differentiation defect of SEC23B null HUDEP2 cells undergoing differentiation. We next generated 7 clonal cell lines with combined deletion for ZBTB7A and SEC23B and confirmed that these cells exhibited normal growth and differentiation indistinguishable from wildtype HUDEP2 cells. The rescuing effect of targeting ZBTB7A was also validated in SEC23B mutated human CD34+ hematopoietic stem and progenitor cell culture. Since SEC23A can functionally replace SEC23B in erythroid cells, we quantified the SEC23A mRNA level in SEC23B-null HUDEP2 cells deleted for ZBTB7A, by qRT-PCR. In early preliminary results, deletion of ZBTB7A resulted in a profound (~10 fold) increase in SEC23A mRNA expression, to a level predicted to be sufficient to rescue the SEC23B null erythroid differentiation defect. ChIP-seq analysis demonstrated that ZBTB7A occupies the SEC23A promoter in HUDEP2 cells. Taken together, these data suggest that ZBTB7A represses SEC23A expression during erythroid maturation and that in the setting of ZBTB7A deletion, SEC23A expression is de-repressed, resulting in rescue of the CDA-II erythroid differentiation defect. Genome editing of the ZBTB7A binding sites in the SEC23A promoter is ongoing in SEC23B-null HUDEP-2 cells. We will determine the impact of the latter editing on SEC23A expression and erythroid differentiation.