Patient-derived Cell Lines Research Articles

Dual MAPK Inhibition Triggers Pro-inflammatory Signals and Sensitizes BRAF V600E Glioma to T Cell-Mediated Checkpoint Therapy.

BRAF V600E pediatric low-grade gliomas frequently transform into high-grade gliomas (HGG) and poorly respond to chemotherapy, resulting in high mortality. Although combined BRAF and MEK inhibition (BRAFi+MEKi) outperforms chemotherapy, ∼70% of BRAF V600E HGG patients are therapy resistant and undergo unbridled tumor progression. BRAF V600E glioma have an immune-rich microenvironment suggesting that they could be responsive to immunotherapy but effects of BRAFi+MEKi on anti-tumor immunity are unclear. Using patient tumor tissue before and after BRAFi+MEKi, two novel syngeneic murine models of BRAF V600E HGG, and patient-derived cell lines, we examined the effects of clinically relevant BRAFi+MEKi with dabrafenib and trametinib on tumor growth, cell states, and tumor-infiltrating T cells. We find that BRAFi+MEKi treatment: i) upregulated programmed cell death protein-1 (PD-1) signaling genes and PD-1 ligand (PD-L1) protein expression in murine BRAF V600E HGG by stimulating IFNγ and IL-27, ii) attenuated T cell activity by IL-23, IL-27 and IL-32 production, which can promote the expansion of regulatory T cells, and iii) induced glial differentiation linked to a therapy-resistant PD-L1+ compartment through Galectin-3 secretion by tumor cells. Murine BRAF V600E HGG shrinkage by BRAFi+MEKi is associated with the upregulation of interferon-gamma response genes, MHC class I/II expression, and antigen presentation and processing programs, indicative of increased anti-tumor immunity. Combined BRAFi+MEKi with therapeutic antibodies inhibiting the PD-1 and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) immune checkpoints re-activate T cells and provide a survival benefit over single therapy in a T cell-dependent manner. The quadruple treatment overcame BRAFi+MEKi resistance by invigorating T cell-mediated anti-tumor immunity in murine BRAF V600E HGG. PD-L1 expression was elevated in human BRAF-mutant versus BRAF-wildtype glioblastoma clinical specimen, complementing experimental findings and suggesting translational relevance for patient care.

Quantitative evaluation of DNA damage repair dynamics to elucidate predictors of autism vs. cancer in individuals with germline PTEN variants.

Persons with germline variants in the tumor suppressor gene phosphatase and tensin homolog, PTEN, are molecularly diagnosed with PTEN hamartoma tumor syndrome (PHTS). PHTS confers high risks of specific malignancies, and up to 23% of the patients are diagnosed with autism spectrum disorder (ASD) and/or developmental delay (DD). The accurate prediction of these two seemingly disparate phenotypes (cancer vs. ASD/DD) for PHTS at the individual level remains elusive despite the available statistical prevalence of specific phenotypes of the syndrome at the population level. The pleiotropy of the syndrome may, in part, be due to the alterations of the key multi-functions of PTEN. Maintenance of genome integrity is one of the key biological functions of PTEN, but no integrative studies have been conducted to quantify the DNA damage response (DDR) in individuals with PHTS and to relate to phenotypes and genotypes. In this study, we used 43 PHTS patient-derived lymphoblastoid cell lines (LCLs) to investigate the associations between DDR and PTEN genotypes and/or clinical phenotypes ASD/DD vs. cancer. The dynamics of DDR of γ-irradiated LCLs were analyzed using the exponential decay mathematical model to fit temporal changes in γH2AX levels which report the degree of DNA damage. We found that PTEN nonsense variants are associated with less efficient DNA damage repair ability resulting in higher DNA damage levels at 24 hours after irradiation compared to PTEN missense variants. Regarding PHTS phenotypes, LCLs from PHTS individuals with ASD/DD showed faster DNA damage repairing rate than those from patients without ASD/DD or cancer. We also applied the reaction-diffusion partial differential equation (PDE) mathematical model, a cell growth model with a DNA damage term, to accurately describe the DDR process in the LCLs. For each LCL, we can derive parameters of the PDE. Then we averaged the numerical results by PHTS phenotypes. By performing simple subtraction of two subgroup average results, we found that PHTS-ASD/DD is associated with higher live cell density at lower DNA damage level but lower cell density level at higher DNA damage level compared to LCLs from individuals with PHTS-cancer and PHTS-neither.

Patient-Derived Cell Line as an In Vitro Model System for Autoantigen-Specific B Cells in Myeloperoxidase-ANCA

Patient-Derived Cell Line as an In Vitro Model System for Autoantigen-Specific B Cells in Myeloperoxidase-ANCA

Single-cell transcriptional atlas of human breast cancers and model systems.

Breast cancer's complex transcriptional landscape requires an improved understanding of cellular diversity to identify effective treatments. The study of genetic variations among breast cancer subtypes at single-cell resolution has potential to deepen our insights into cancer progression. In this study, we amalgamate single-cell RNA sequencing data from patient tumours and matched lymph metastasis, reduction mammoplasties, breast cancer patient-derived xenografts (PDXs), PDX-derived organoids (PDXOs), and cell lines resulting in a diverse dataset of 117 samples with 506719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 positive (HER2+), and triple-negative breast cancer (TNBC) subtypes, including isogenic model pairs. Herein, we delineated similarities and distinctions across models and patient samples and explore therapeutic drug efficacy based on subtype proportions. PDX models more closely resemble patient samples in terms of tumour heterogeneity and cell cycle characteristics when compared with TNBC cell lines. Acquired drug resistance was associated with an increase in basal-like cell proportions within TNBC PDX tumours as defined with SCSubtype and TNBCtype cell typing predictors. All patient samples contained a mixture of subtypes; compared to primary tumours HR+ lymph node metastases had lower proportions of HER2-Enriched cells. PDXOs exhibited differences in metabolic-related transcripts compared to PDX tumours. Correlative analyses of cytotoxic drugs on PDX cells identified therapeutic efficacy was based on subtype proportion. We present a substantial multimodel dataset, a dynamic approach to cell-wise sample annotation, and a comprehensive interrogation of models within systems of human breast cancer. This analysis and reference will facilitate informed decision-making in preclinical research and therapeutic development through its elucidation of model limitations, subtype-specific insights and novel targetable pathways. Patient-derived xenografts models more closely resemble patient samples in tumour heterogeneity and cell cycle characteristics when compared with cell lines. 3D organoid models exhibit differences in metabolic profiles compared to their in vivo counterparts. A valuable multimodel reference dataset that can be useful in elucidating model differences and novel targetable pathways.

Blockade of Src signaling prevented stemness gene expression and proliferation of patient-derived gastric cancer stem cells

ABSTRACT Objectives: Gastric cancer (GC) is one of the most malignant tumors. Mounting studies highlighted gastric cancer stem cells (GCSCs) were responsible for the failure of treatment due to recurrence and drug resistance of advanced GC. However, targeted therapy against GCSC for improving GC prognosis suffered from lack of suitable models and molecular targets in terms of personalized medicine. To address this issue, two patient-derived GC cell lines SD209 and SD292 with cancer stem cells (CSCs) such as phenotype were isolated for establishing targeted therapy aiming at critical metastatic signaling in GC. Materials and Methods: The primary patient-derived GCSCs were established from parts of GC tissues for characterization of stem cells (SCs) phenotype at both cellular and molecular levels. Western blot and Immunohistochemistry (IHC) were performed for identifying the deregulated signaling in GC tissue. Immunofluorescence was used for analyzing proliferating and SC markers in GCSC attached on fibroblast. Acridine orange and propidium iodide analyses were performed for the survival of GCSC in suspensions. Results: In the culture environments of both SD209 and SD292, a lot of mesenchymal fibroblasts spread and crowd together on which a lot of cell clumps, suspected as GCSC, were firmly attached. In the IHC analysis, the GCSC stemness genes CD44 and Ep-CAM increased in tumor tissues of SD209, whereas Nanog-1 and octamer-binding transcription factor 3 (OCT-3) increased in that of SD292. By immunofluorescent analysis of a proliferation marker Ki67, the growth of SD209 and SD292 on mesenchymal fibroblasts was found to be reduced by dasatinib, the inhibitor of the Src kinase whose activity was upregulated in tumor tissues of both GCs. Dasatinib also suppressed the expression of Nanog-1 and OCT-3 in SD292 attached on mesenchymal fibroblasts. Conclusion: This study may provide a base for targeted therapy against GCSCs/GCs progression in future preclinical/clinical settings.

Impact of Proton Irradiation Depending on Breast Cancer Subtype in Patient-Derived Cell Lines

Research on different types of ionizing radiation’s effects has been ongoing for years, revealing its efficacy in damaging cancer cells. Solid tumors comprise diverse cell types, each being able to respond differently to radiation. This study evaluated the radiobiological response of established (MDA-MB-231 (Triple negative breast cancer, TNBC), MCF-7 (Luminal A)) and patient-derived malignant cell lines, cancer-associated fibroblasts, and skin fibroblasts following proton IRR. All cell line types were irradiated with the proton dose of 2, 4, and 6 Gy. The radiobiological response was assessed using clonogenic assay, γH2AX, and p53 staining. It was noticeable that breast cancer lines of different molecular subtypes displayed no significant variations in their response to proton IRR. In terms of cancer-associated fibroblasts extracted from the tumor tissue, the line derived from a TNBC subtype tumor demonstrated higher resistance to ionizing radiation compared to lines isolated from luminal A tumors. Fibroblasts extracted from patients’ skin responded identically to all doses of proton radiation. This study emphasizes that tumor response is not exclusively determined by the elimination of breast cancer cells, but also takes into account tumor microenvironmental variables and skin reactions.

Targeting RNA helicase DDX3X with a small molecule inhibitor for breast cancer bone metastasis treatment

Patients who present with breast cancer bone metastasis only have limited palliative treatment strategies and efficacious drug treatments are needed. In breast cancer patient data, high levels of the RNA helicase DDX3 are associated with poor overall survival and bone metastasis. Consequently, our objective was to target DDX3 in a mouse breast cancer bone metastasis model using a small molecule inhibitor of DDX3, RK-33. Histologically confirmed live imaging indicated no bone metastases in the RK-33 treated cohort, as opposed to placebo-treated mice. We generated a cell line from a bone metastatic lesion in mouse and found that it along with a patient-derived bone metastasis cell line gained resistance to conventional chemotherapeutics but not to RK-33. Finally, differential levels of DDX3 were observed in breast cancer patient metastatic bone samples. Overall, this study indicates that DDX3 is a relevant clinical target in breast cancer bone metastasis and that RK-33 can be a safe and effective treatment for these patients.

High-throughput drug screening identifies novel therapeutics for Low Grade Serous Ovarian Carcinoma

Low grade serous carcinoma (LGSOC) is a rare epithelial ovarian cancer with unique molecular characteristics compared to the more common tubo-ovarian high-grade serous ovarian carcinoma. Pivotal clinical trials guiding the management of epithelial ovarian cancer lack sufficient cases of LGSOC for meaningful subgroup analysis, hence overall findings cannot be extrapolated to rarer chemo-resistant subtypes such as LGSOC. Furthermore, there is a need for more effective therapies for the treatment of relapsed disease, as treatment options are limited. To address this, we conducted the largest quantitative high-throughput drug screening effort (n = 3436 compounds) in 12 patient-derived LGSOC cell lines and one normal ovary cell line to identify unexplored therapeutic avenues. Using a combination of high-throughput robotics, high-content imaging and novel data analysis pipelines, our data set identified 60 high and 19 moderate confidence hits which induced cancer cell specific cytotoxicity at the lowest compound dose assessed (0.1 µM). We also revealed a series of known (mTOR/PI3K/AKT) and novel (EGFR and MDM2-p53) drug classes in which LGSOC cell lines showed demonstrable susceptibility to.

A deep intronic splice-altering AIRE variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendritic cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE-expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

Polyamine Pathway Inhibitor DENSPM Suppresses Lipid Metabolism in Pheochromocytoma Cell Line.

Pheochromocytomas (PCCs) are tumors arising from chromaffin cells in the adrenal medulla, and paragangliomas (PGLs) are tumors derived from extra-adrenal sympathetic or parasympathetic paraganglia; these tumors are collectively referred to as PPGL cancer. Treatment for PPGL primarily involves surgical removal of the tumor, and only limited options are available for treatment of the disease once it becomes metastatic. Human carriers of the heterozygous mutations in the succinate dehydrogenase subunit B (SDHB) gene are susceptible to the development of PPGL. A physiologically relevant PCC patient-derived cell line hPheo1 was developed, and SDHB_KD cells carrying a stable short hairpin knockdown of SDHB were derived from it. An untargeted metabolomic approach uncovered an overactive polyamine pathway in the SDHB_KD cells that was subsequently fully validated in a large set of human SDHB-mutant PPGL tumor samples. We previously reported that treatment with the polyamine metabolism inhibitor N1,N11-diethylnorspermine (DENSPM) drastically inhibited growth of these PCC-derived cells in culture as well as in xenograft mouse models. Here we explored the mechanisms underlying DENSPM action in hPheo1 and SDHB_KD cells. Specifically, by performing an RNAseq analysis, we have identified gene expression changes associated with DENSPM treatment that broadly interfere with all aspects of lipid metabolism, including fatty acid (FA) synthesis, desaturation, and import/uptake. Furthermore, by performing an untargeted lipidomic liquid chromatography-mass spectrometry (LC/MS)-based analysis we uncovered specific groups of lipids that are dramatically reduced as a result of DENSPM treatment. Specifically, the bulk of plasmanyl ether lipid species that have been recently reported as the major determinants of cancer cell fate are notably decreased. In summary, this work suggests an intersection between active polyamine and lipid pathways in PCC cells.

Abstract A067: Identifying and targeting FOLFIRINOX resistance mechanisms to improve outcomes in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of only 12%. More than 80% of PDAC patients cannot undergo potentially curative surgical resection due to the presentation of advanced, metastatic disease, and are instead offered systemic chemotherapy regimens to which resistance is rapidly acquired. FOLFIRINOX chemotherapy offers an 11.1-month median survival compared to 6.8 months with Gemcitabine. Rapid onset of resistance to FOLFIRINOX, coupled with the lack of alternative targeted therapies, ultimately results in patients succumbing to the disease. My project aims to identify mechanisms by which PDAC cells acquire resistance to FOLFIRINOX treatment and investigate interventions aimed at enhancing and/or reinstating FOLFIRINOX sensitivity. Our lab has propagated distinct patient derived xenografts (PDXs) from the Avner Australian Pancreatic Cancer Matrix Atlas (APMA) and I have exposed them to 11 - 15 rounds of FOLFIRINOX (or vehicle control) in vivo to reflect the clinical management of PDAC. Additionally, intrapancreatic orthotopic tumors derived from cells from the KPC mouse model (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) have received 5 - 6 rounds of FOLFIRINOX (or vehicle control). These tumors will undergo quantitative data-independent acquisition mass spectrometry proteomic analysis and RNA sequencing transcriptomic analysis (RNAseq) to reveal resistance pathways and/or proteins operating in the chronically treated tumors and the surrounding pancreatic tumor microenvironment. Additionally, we have developed and cultured matched treatment naïve and FOLFIRINOX-treated patient-derived cell lines (PDCLs) and mouse cell lines from the PDX and KPC-derived tumors. These lines will allow us to validate future targets via gene knockdown and/or pharmacological intervention. 3D assays, such as our organotypic matrix models, will be performed to assess the metastatic potential of cancer cells and their response to FOLFIRINOX in vitro. Furthermore, subcutaneous and orthotopic (intrapancreatic) models coupled with intravital (in vivo) imaging and biosensor technology will allow us to assess targeting in live tumors and metastatic sites in real time. It is the hope that this research will lead to the identification of proteins or protein pathways which can be manipulated in order to re-sensitize tumors to FOLFIRINOX chemotherapy. Citation Format: Victoria Tyma, Ying-Fei Liew, Alice Tran, Anna Howell, Shona Ritchie, Avner Australian Pancreatic Cancer Matrix Atlas, Australian Pancreatic Genome Initiative, Thomas Cox, Marina Pajic, David Herrmann, Brooke Pereira, Paul Timpson, Katie Gordon. Identifying and targeting FOLFIRINOX resistance mechanisms to improve outcomes in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A067.

Abstract A079: SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, with only 12% of patients living longer than five years after diagnosis. Recent studies have shown that inhibiting microtubule dynamics may be a promising therapeutic avenue for treating patients with this cancer. Microtubules are critical in cancer cell growth and division, as well as intracellular cytoskeletal support and internal trafficking. In PDAC, βIII- and βIVb-tubulin are highly upregulated in contrast to normal pancreas cells. Decreased expression of βIII- and βIVb-tubulin, through silencing or inhibition, correlates with reduced PDAC cell growth, tumorigenic potential, metastases in xenograft models, and chemoresistance. We hypothesized that a novel colchicine binding site beta-tubulin inhibitor, SB-216, will decrease cell growth while also decreasing the expression of oncogenic βIII- and βIVb-tubulin. To examine the anti-proliferative effects, PDCL110, a PDAC patient-derived cell line, cells were seeded (6,000 cells/well), and after 24 hours, treated with 0.1% DMSO (control), 2 nM, 5 nM, 10 nM, or 15 nM SB-216. Plates were analyzed on the IncuCyte Live-Cell Analysis System for six days, where phase contrast images of confluency were captured every four hours. To characterize the mRNA expression of βIII- and βIVb-tubulin, Panc-1 and PDCL110 cells were treated with 0.1% DMSO and 5 nM SB-216 for 48 hours. Next, total RNA was extracted using a TRIzol reagent. cDNAs were prepared using the SYBR Green RNA Reverse Transcription kit, and mRNA expression of beta-tubulin isotypes was quantified using the QuantStudio Real-Time PCR system. The expression of tubulin isotypes was normalized with the internal control ACTIN. Cell growth curves illustrated a significant (p<0.006) decrease in proliferation after 48 hours of treatment at all tested concentrations of SB-216. After 72 hours, cell growth inhibition became more significant (p<0.0001) in a dose-dependent manner. SB-216 treatment of a commercially available PDAC cell line (Panc-1) led to decreased mRNA expression of TUBB3 (class βIII, p<0.05) and TUBB4B (class βIVb, p<0.05) compared to DMSO control. Similarly, TUBB4B expression was significantly (p<0.05) decreased in the PDCL110 cells upon SB-216 treatment. These results enhance our confidence in utilizing SB-216 as a therapeutic agent against PDAC. Future studies are directed at elucidating the effects of SB-216 on metastases, chemoresistance, and mitochondrial function. Through an improved understanding of the mechanisms behind microtubule inhibition, we can meet the urgent clinical need of finding targeted treatments for this complex cancer. Citation Format: Lauren C Gattie, Chun Cai, Rui Wang, Wei Li, Evan Glazer. SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A079.

Novel potent molecular glue degraders against broad range of hematological cancer cell lines via multiple neosubstrates degradation

BackgroundTargeted protein degradation of neosubstrates plays a crucial role in hematological cancer treatment involving immunomodulatory imide drugs (IMiDs) therapy. Nevertheless, the persistence of inevitable drug resistance and hematological toxicities represents a significant obstacle to their clinical effectiveness.MethodsPhenotypic profiling of a small molecule compounds library in multiple hematological cancer cell lines was conducted to screen for hit degraders. Molecular dynamic-based rational design and cell-based functional assays were conducted to develop more potent degraders. Multiple myeloma (MM) tumor xenograft models were employed to investigate the antitumor efficacy of the degraders as single or combined agents with standard of care agents. Unbiased proteomics was employed to identify multiple therapeutically relevant neosubstrates targeted by the degraders. MM patient-derived cell lines (PDCs) and a panel of solid cancer cell lines were utilized to investigate the effects of candidate degrader on different stage of MM cells and solid malignancies. Unbiased proteomics of IMiDs-resistant MM cells, cell-based functional assays and RT-PCR analysis of clinical MM specimens were utilized to explore the role of BRD9 associated with IMiDs resistance and MM progression.ResultsWe identified a novel cereblon (CRBN)-dependent lead degrader with phthalazinone scaffold, MGD-4, which induced the degradation of Ikaros proteins. We further developed a novel potent candidate, MGD-28, significantly inhibited the growth of hematological cancer cells and induced the degradation of IKZF1/2/3 and CK1α with nanomolar potency via a Cullin-CRBN dependent pathway. Oral administration of MGD-4 and MGD-28 effectively inhibited MM tumor growth and exhibited significant synergistic effects with standard of care agents. MGD-28 exhibited preferentially profound cytotoxicity towards MM PDCs at different disease stages and broad antiproliferative activity in multiple solid malignancies. BRD9 modulated IMiDs resistance, and the expression of BRD9 was significant positively correlated with IKZF1/2/3 and CK1α in MM specimens at different stages. We also observed pronounced synergetic efficacy between the BRD9 inhibitor and MGD-28 for MM treatment.ConclusionsOur findings present a strategy for the multi-targeted degradation of Ikaros proteins and CK1α against hematological cancers, which may be expanded to additional targets and indications. This strategy may enhance efficacy treatment against multiple hematological cancers and solid tumors.

Epigenetic therapy potentiates transposable element transcription to create tumor-enriched antigens in glioblastoma cells.

Inhibiting epigenetic modulators can transcriptionally reactivate transposable elements (TEs). These TE transcripts often generate unique peptides that can serve as immunogenic antigens for immunotherapy. Here, we ask whether TEs activated by epigenetic therapy could appreciably increase the antigen repertoire in glioblastoma, an aggressive brain cancer with low mutation and neoantigen burden. We treated patient-derived primary glioblastoma stem cell lines, an astrocyte cell line and primary fibroblast cell lines with epigenetic drugs, and identified treatment-induced, TE-derived transcripts that are preferentially expressed in cancer cells. We verified that these transcripts could produce human leukocyte antigen class I-presented antigens using liquid chromatography with tandem mass spectrometry pulldown experiments. Importantly, many TEs were also transcribed, even in proliferating nontumor cell lines, after epigenetic therapy, which suggests that targeted strategies like CRISPR-mediated activation could minimize potential side effects of activating unwanted genomic regions. The results highlight both the need for caution and the promise of future translational efforts in harnessing treatment-induced TE-derived antigens for targeted immunotherapy.

Introduction of an RS1 mutation causative variant consistent with identified XLRS patient using CRISPR/Cas9 technology in normal iPSC

X-linked retinoschisis (XLRS) is a common retinal genetic disease that occurs in juvenile males and causes progressive visual impairment. this presents a schisis in the macula or peripheral retina of bilateral eyes and currently has no effective treatment. Here, we introduced the RS1 (c.C304T, p.R102W) mutation into a normal induced pluripotent stem (iPS) cell line using CRISPR/Cas9 technology. This missense mutation was consistent with that observed in the XLRS patient-derived iPS cell line (CSUASOi001-A). Conclusively, establishing a directed gene mutation cell line (CSUi007-A) provides a useful cell resource to investigate XLRS pathogenesis.

Decoding Endothelial MPL and JAK2V617F Mutation: Insight Into Cardiovascular Dysfunction in Myeloproliferative Neoplasms.

Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential face a significantly elevated risk of cardiovascular diseases. Endothelial cells carrying the JAK2V617F mutation have been detected in many patients with MPN. In this study, we investigated the molecular basis for the high incidence of cardiovascular complications in patients with MPN. We investigated the impact of endothelial JAK2V617F mutation on cardiovascular disease development using both transgenic murine models and MPN patient-derived induced pluripotent stem cell lines. Our investigations revealed that JAK2V617F mutant endothelial cells promote cardiovascular diseases under stress, which is associated with endothelial-to-mesenchymal transition and endothelial dysfunction. Importantly, we discovered that inhibiting the endothelial TPO (thrombopoietin) receptor MPL (myeloproliferative leukemia virus oncogene) suppressed JAK2V617F-induced endothelial-to-mesenchymal transition and prevented cardiovascular dysfunction caused by mutant endothelial cells. Notably, the endothelial MPL receptor is not essential for the normal physiological regulation of blood cell counts and cardiac function. JAK2V617F mutant endothelial cells play a critical role in the development of cardiovascular diseases in JAK2V617F-positive MPNs, and endothelial MPL could be a promising therapeutic target for preventing or ameliorating cardiovascular complications in these patients.

Establishment and characterization of two novel patient-derived cell lines from myxofibrosarcoma: NCC-MFS7-C1 and NCC-MFS8-C1.

Myxofibrosarcoma (MFS), an aggressive soft tissue sarcoma, presents a significant challenge because of its high recurrence rate, distal metastasis, and complex genetic background. Although surgical resection is the standard treatment for MFS, the outcomes are unsatisfactory and effective non-surgical treatment strategies, including drug therapy, are urgently warranted. MFS is a rare tumor that requires comprehensive preclinical research to develop promising drug therapies; however, only two MFS cell lines are publicly available worldwide. The present study reports two novel patient-derived MFS cell lines, NCC-MFS7-C1 and NCC-MFS8-C1. These cell lines have been extensively characterized for their genetic profile, proliferation, spheroid-forming capacity, and invasive behavior, confirming that they retain MFS hallmarks. Furthermore, we conducted comprehensive drug screening against these cell lines and six others previously established in our laboratory to identify potential therapeutic candidates for MFS. Among the screened agents, actinomycin D, bortezomib, and romidepsin demonstrated considerable antiproliferative effects that were superior to those of doxorubicin, a standard drug, highlighting their potential as novel drugs. In conclusion, NCC-MFS7-C1 and NCC-MFS8-C1 are valuable research resources that contribute to the understanding of the pathogenesis and development of novel therapies for MFS.

A cell state-specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma

Glioma cells hijack developmental programs to control cell state. Here, we uncover a glioma cell state-specific metabolic liability that can be therapeutically targeted. To model cell conditions at brain tumor inception, we generated genetically engineered murine gliomas, with deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling astrocyte differentiation during brain development. N1IC tumors harbored quiescent astrocyte-like transformed cell populations while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. Further, N1IC transformed cells exhibited increased mitochondrial lipid peroxidation, high ROS production and depletion of reduced glutathione. This altered mitochondrial phenotype rendered the astrocyte-like, quiescent populations more sensitive to pharmacologic or genetic inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Treatment of patient-derived early-passage cell lines and glioma slice cultures generated from surgical samples with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles. Collectively, these findings reveal a specific therapeutic vulnerability to ferroptosis linked to mitochondrial redox imbalance in a subpopulation of quiescent astrocyte-like glioma cells resistant to standard forms of treatment.

Establishment and characterization of NCC-SS6-C1: a novel patient-derived cell line of synovial sarcoma.

Synovial sarcoma (SS) is identified as a sarcoma with monomorphic blue spindle cells that display variable epithelial differentiation and is characterized by the SS18::SSX fusion gene. SS accounts for approximately 5-10% of all soft tissue sarcomas, making it a relatively common type within this group of tumors. Since SS is generally sensitive to chemotherapy, the standard treatment for SS includes extensive surgical resection, complemented by neoadjuvant chemotherapy with several approved anticancer drugs. However, in advanced and metastatic cases, the efficacy of these drugs is limited, resulting in poor prognoses. This underscores the need for innovative therapeutic strategies. Patient-derived cancer cell lines are essential tools for basic and preclinical research, yet only four SS cell lines are publicly available. To facilitate the studies of SS, we have developed a novel SS cell line, named NCC-SS6-C1, derived from surgically excised tumor tissue of an SS patient. NCC-SS6-C1 cells preserve the SS18::SSX1 fusion gene, consistent with the genetic characteristics of the original tumor. The cells exhibit continuous proliferation, invasiveness, and the ability to form spheroids. Additionally, we confirmed that this cell line was useful for evaluating the efficacy of anticancer drugs. Our results suggest that NCC-SS6-C1 is a useful tool for basic and pre-clinical studies of SS.

Generation of hereditary spastic paraplegia patient-derived induced pluripotent stem cell line UJSi003-A

Hereditary spastic paraplegia (HSP) is a rare neurodegenerative disorder with the predominant clinical manifestation of spasticity in the lower extremities. Patients with HSP experience spastic paralysis in both lower limbs, leading to progressive walking difficulties, increased reflexes, spasms, and extensor plantar responses. We successfully generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with HSP. The iPSCs exhibited a normal karyotype, expressed pluripotency markers, and differentiated into the three germ layers in vitro.