Patient-derived Tumor Cells Research Articles

Establishment of Patient-Derived Organoids Using Ascitic or Pleural Fluid from Cancer Patients.

Patient-derived tumor cells can be a powerful resource for studying pathophysiological mechanisms and developing robust strategies for precision medicine. However, establishing organoids from patient-derived cells is challenging because of limited access to tissue specimens. Therefore, we aimed to establish organoids from malignant ascites and pleural effusions. Ascitic or pleural fluid from pancreatic, gastric, and breast cancer patients was collected and concentrated to culture tumor cells ex vivo. Organoids were considered to be successfully cultured when maintained for five or more passages. Immunohistochemical staining was performed to compare the molecular features, and drug sensitivity was assayed to analyze the clinical responses of original patients. We collected 70 fluid samples from 58 patients (pancreatic cancer, n=39; gastric cancer, n=21; and breast cancer, n=10). The overall success rate was 40%; however, it differed with types of malignancy, with pancreatic, gastric, and breast cancers showing 48.7%, 33.3%, and 20%, respectively. Cytopathological results significantly differed between successful and failed cases (p=0.014). Immunohistochemical staining of breast cancer organoids showed molecular features identical to those of tumor tissues. In drug sensitivity assays, pancreatic cancer organoids recapitulated the clinical responses of the original patients. Tumor organoids established from malignant ascites or pleural effusion of pancreatic, gastric, and breast cancers reflect the molecular characteristics and drug sensitivity profiles. Our organoid platform could be used as a testbed for patients with pleural and peritoneal metastases to guide precision oncology and drug discovery.

Measurement of Patient-Derived Glioblastoma Cell Response to Temozolomide Using Fluorescence Lifetime Imaging of NAD(P)H.

Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α2 associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response-cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients.

Sensitive, High-Throughput HLA-I and HLA-II Immunopeptidomics Using Parallel Accumulation-Serial Fragmentation Mass Spectrometry

Comprehensive and in-depth identification of the human leukocyte antigen class I (HLA-I) and class II (HLA-II) tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is a powerful technology for direct identification of HLA peptides from patient-derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare and clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high-throughput, sensitive, and single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight MS on the Bruker timsTOF single-cell proteomics system (SCP). We demonstrate greater than twofold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation, and reduces input requirements to as few as 1e6 A375cells for >800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen and noncanonical proteins. We also apply our optimized single-shot SCP acquisition methods to tumor-derived samples, enabling sensitive, high-throughput, and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15mg wet weight tissue.

A novel CDK4/6 inhibitor combined with irradiation demonstrates potent anti-tumor efficacy in diffuse midline glioma

Diffuse midline glioma, H3 K27-altered (DMG) is a lethal pediatric brainstem tumor. Despite numerous efforts to improve survival benefits, its prognosis remains poor. This study aimed to design and synthesize a novel CDK4/6 inhibitor YF-PRJ8-1011, which exhibited more potent antitumor activity against a panel of patient-derived DMG tumor cells in vitro and in vivo compared with palbociclib. Patient-derived DMG cells were used to assess the antitumor efficacy of YF-PRJ8-1011 in vitro. The liquid chromatography tandem-mass spectrometry method was used to measure the activity of YF-PRJ8-1011 passing through the blood-brain barrier. DMG patient-derived xenograft models were established to detect the antitumor efficacy of YF-PRJ8-1011. The results showed that YF-PRJ8-1011 could inhibit the growth of DMG cells both in vitro and in vivo. YF-PRJ8-1011 could well penetrate the blood-brain barrier. It also significantly inhibited the growth of DMG tumors and prolonged the overall survival of mice compared with vehicle or palbociclib. Most notably, it exerted potent antitumor efficacy in DMG in vitro and in vivo compared with palbociclib. In addition, we also found that YF-PRJ8-1011 combined with radiotherapy also showed more significant inhibition of DMG xenograft tumor growth than radiotherapy alone. Collectively, YF-PRJ8-1011 is a novel, safe, and selective CDK4/6 inhibitor for DMG treatment.

Ketorolac modulates Rac-1/HIF-1α/DDX3/β-catenin signalling via a tumor suppressor prostate apoptosis response-4 (Par-4) in renal cell carcinoma

Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20–30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cell-cycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac’s impact on metastasis by influencing Rac-1/HIF-1α/DDX3/β-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/Cdc42/HIF-1α/DDX3/β-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC.

Abstract 4486: Proof of principle for pharmacogenomic-guided precision oncology for pediatric malignancy

Abstract Childhood cancers are rare and clinically diverse. They are typically associated with few driver mutational events than adult cancers, constituting potential targets for patient-specific precision therapy approaches. Here, we evaluated the feasibility of using a patient-derived tumor cell (PDC) based drug screening system and integrated multi-omics data to achieve precision oncology for pediatric patients. We established a PDC library derived from a few passage-cultured tumor cells from surgically resected tumor specimens and conducted chemical screening, which composed of various target agents of major oncogenic pathways (e.g. receptor tyrosine kinase inhibitor, proteasome inhibitor and histone deacetylase etc.). Next Generation Sequencing was performed to characterize the genomic and transcriptomic traits of tumors. Overall, success of establishing PDCs of pediatric cancers was high (80.4%) and comparable to adult cancers. The amount of obtained tissue was an important factor for the success of PDC establishment; the estimated optimal weight for PDC establishment was small (1.14g) and it is noteworthy that a small amount of tumor sample is sufficient to identify the potential hit(s) of parental tumors. The platform provided therapeutic options to pediatric tumors regardless of actionable targets. Our PDC approach identified several potential gene-drug associations, including anti-PI3K/Akt agents for neuroblastomas and anti-SHH agents for sarcomas with EWSR1 fusion. Given that a considerable proportion of pediatric tumors still lack actionable targets with matched treatment options, PDC-based drug screening profiles can be an optimal therapeutic strategy for these cases. Collectively, our analysis confirmed the feasibility of PDC-based in vitro drug screening systems to guide the therapeutic strategy for pediatric patients. This approach will accelerate the preclinical research for pediatric tumors to understand their pathophysiology and investigate the potential therapeutic strategies to fulfill the future precision oncology. Citation Format: Gi Ju Lee, Seung-Won Choi, Seung Ah Choi, Hee-Jin Cho, Robyn Gartrell, Ji Won Lee, Joo Whan Kim, Nam-Gu Her, Raul Rabadan, Ki Woong Sung, Do-Hyun Nam, Seung-Ki Kim. Proof of principle for pharmacogenomic-guided precision oncology for pediatric malignancy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4486.

Abstract 161: Patient-derived organoids (PDOs) hub of National Cancer Center, Korea: pre-clinical model for drug screening

Abstract Purpose Cancer is one of the leading causes of death worldwide. Patient-derived tumor cells can serve as a powerful resource for studying pathophysiologic mechanisms and developing robust strategies for precision medicine. To address this problem, we launched the patient-derived organoids (PDOs) Hub to establish a comprehensive model of various tumor organoids from pancreatic, biliary tract, liver, colorectal, breast, gastric, ovarian, and oral cancers, with matching clinical data and molecular characteristics. Methods All specimens were collected from histologically confirmed cancer patients at the National Cancer Center. Samples obtained from surgery, biopsy, or body fluid (malignant ascites or pleural effusion) were collected for ex vivo culture of tumor cells. PDOs were managed according to our standard operating procedure (SOP), which included specimen delivery process, separation of cells from tissues, criteria for subculture, quality control (QC), production of genomic and histologic data, and the 384-well-based drug response evaluation system. Organoids were considered to be successfully cultured when they were maintained for five or more passages. Results A total of 263 PDOs were established from various cancer types, including oral cancer (N = 89), pancreatic cancer (N = 48), ovarian cancer (N = 32), breast cancer (N = 30), biliary tract cancer (N = 29), hepatocellular carcinoma (N = 17), gallbladder cancer (N = 8), gastric cancer (N = 7) and colorectal cancer (N = 3). PDOs broadly recapitulated the histologic and genetic characteristics of the patient’s tumor. These organoids available for long-term culture were cryopreserved, and a total of 2986 stocks have been accumulated. Drug screening tests were performed with 60 PDOs (pancreatic cancer, N = 36; breast cancer, N = 15; ovarian cancer, N = 6; gastric cancer, N = 3) using selected agents among the 47 drugs for each type of cancer. Profiles of cytotoxic agents were well correlated with the patient’s clinical responses to the matched drugs and tested investigational agents also showed promising antitumor activity. Conclusions We have established a model of several human cancer organoids. This will serve as the platform that can recapitulate the physiology and drug response profiles of human cancer and pave the way for screening innovative drugs, identifying novel targets, and stratifying patients for pertinent therapeutic options. (This work was supported by National Research Foundation of Korea grant, funded by the Korean government (MSIT) (No. 2020M3A9A5036362)) Citation Format: Yebeen Yu, Mi Rim Lee, Wonyoung Choi, Sumin Kang, Jeong Eun Gong, Soobeen Heo, Hye Ju Park, Sang Myung Woo, So-Youn Jung, Sung Weon Choi, Jong-Ho Lee, Myong Cheol Lim, Ji Yeon Baek, Bo Hyun Kim, Ji Hoon Kim, Yuri Cho, Sang-Jae Park, Yun-Hee Kim, Sun-Young Kong. Patient-derived organoids (PDOs) hub of National Cancer Center, Korea: pre-clinical model for drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 161.

Abstract 4940: Proteasome and survivin inhibitors synergize to inhibit the growth of oral cancer organoids

Abstract Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head and neck cancers that has a disproportionately high incidence and mortality in developing countries in South-Central Asia, including Pakistan. The standard treatment for OSCC generally consists of surgery combined with radiotherapy or chemotherapy; however, the overall survival rates have not improved, and only ~50% of patients survive the disease. Therefore, identifying and validating new drug targets can provide new therapeutic options for improved treatment and better prognosis for OSCC patients. The present study aimed to develop an organoid model from patient-isolated primary tumour cells and determine their drug sensitivity profile coupled with mutational analysis. New combinations of targeted drugs with synergistic activity in oral cancer cells were identified through high-throughput screening followed by validation of their activity in oral cancer cells in 2D monolayer and 3D organoid cultures. Results indicate a synergistic activity of proteasome (PR-171) and survivin (YM155) inhibitors in all tested patient-derived tumour cells and four oral cancer cell lines. Moreover, chitosan-based nanocapsules harboring these drugs showed therapeutic efficacy comparable to free drug combinations, indicating a potential for efficient and targeted delivery. Next-generation sequencing data identified missense mutations in eleven cancer-related genes that encode BRAF, EGFR, KRAS, NRAS, HRAS, MEK1, PIK3CA, PTEN, NOTCH1, TP53 and TERT. The ongoing research aims to correlate the drug sensitivity data with the mutational profile of patient-derived tumour cells to find novel targets. In conclusion, we have developed 3D organoids from patient-derived primary tumours and utilized them to validate the synergistic combination between screen-identified proteasome and survivin inhibitors. Our preclinical study provides a rationale for further evaluation of the novel drug combination in vivo and in clinical settings. Citation Format: Muhammad Furqan, Iffat Aleem, Muhammad Tahseen, Raza Hussain, Asif Loya, Muhammad Tariq Mahmood, Philippe Gautier, Kevin Myant, Saira Saleem, Amir Faisal. Proteasome and survivin inhibitors synergize to inhibit the growth of oral cancer organoids. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4940.

Abstract 4964: SW-682: A novel TEAD inhibitor for the treatment of cancers bearing mutations in the Hippo signaling pathway

Abstract Many cancers harbor mutations in the Hippo pathway that lead to constitutive activation of the transcriptional co-activators YAP/TAZ that then bind the transcription factor TEAD and drive aberrant transcription of target genes involved in cell proliferation and tumor progression. Hyperactivation of YAP/TAZ has also been associated with resistance to targeted therapies, including MAPK pathway inhibitors. To target cancers that bear mutations in the Hippo pathway or are resistant to therapies due to YAP/TAZ activation, we developed SW-682, a pan-TEAD small molecule inhibitor that blocks TEAD-dependent transcription by binding to the palmitoylation pocket of all TEAD isoforms. In vitro, SW-682 inhibited the proliferation of human Hippo-mutant mesothelioma cells with nanomolar potency, with little to no effect on Hippo wild-type tumor cells. SW-682 down-regulated TEAD-dependent reporter gene expression in a dose-dependent manner, while having no effect on reporters monitoring other pathways. In vivo, daily oral administration of SW-682 to adult mice resulted in tumor regression in Hippo-mutant mesothelioma models and caused down-regulation of expression of the TEAD-dependent genes CCN1 and CCN2 and a YAP gene signature, as measured by qPCR or RNA-seq analysis. SW-682 has a favorable PK profile with good oral bioavailability in the mouse and was well tolerated with no signs of body weight loss. To test the hypothesis that TEAD inhibition can overcome YAP-driven resistance mechanisms, we explored SW-682 in combination with MEK inhibitors in several in vitro and ex vivo patient-derived tumor models including BRAF and NRAS mutated melanoma. Moreover, to identify new indications that may benefit from TEAD inhibition, we screened patient-derived 3D organoid tumor cells and matching patient-derived xenograft models that have been molecularly profiled. In summary, SW-682 is a potent and selective investigational TEAD inhibitor which demonstrated anti-tumor effects in models harboring aberrant expression of the Hippo pathway, suggesting therapeutic potential in multiple Hippo-mutant solid tumors. Citation Format: Lei Chen, Paula Milani de Marval, Kendall Powell, Mark Johnson, Greg Falls, Brian Lawhorn, Aurélie Candi, Amuri Kilonda, Bart Vanderhoydonck, Arnaud Marchand, Matthias Versele, Georg Halder, Stephen L. Gwaltney, Adeela Kamal. SW-682: A novel TEAD inhibitor for the treatment of cancers bearing mutations in the Hippo signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4964.

Abstract 36: Comparing twenty-two matched patient-derived cell lines developed from either patient, PDX, or organoid tumor cell material

Abstract The National Cancer Institute has developed a Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) comprised of quality-controlled, early passage, and clinically-annotated patient-derived tumor xenografts (PDXs), organoids (PDOrgs), cell cultures (PDCs), and cancer associated fibroblasts (CAFs) available with genomic data to the extramural community for research use. Models are developed by the NCI PDMR in decreasing order of complexity, specifically 1) patient material can be used to develop PDXs, PDOrgs, PDCs, and CAFs, 2) PDX material for PDOrgs and PDCs, and 3) PDOrg material for PDCs, but a PDC is never used to develop a PDOrg or PDX. Eleven pairs of 22 matched PDCs have been developed in parallel from either patient, PDX, patient-derived organoid, or PDX-derived organoid tumor material and sequenced by WES and RNASeq. Genetic stability was assessed using multiple approaches including microsatellite instability (MSI) generated from MSISensor2, percentage of genomic loss of heterozygosity (LOH) using a set of ~800k heterozygous SNPs from a population level genomic database (gnomAD), pairwise Spearman correlation based on BIN level copy number (CN)/RNA expression profiles, and OncoKB annotated oncogenic/likely oncogenic variants. No systematic differences were observed within PDC pairs derived from different origins or compared to their patient and/or PDX material in MSI, LOH% and RNA expression profile but pairwise Spearman correlation (0.66-0.88) in CNV profiles were somewhat variable, likely due to low sequencing depth. In one PDC pair (299254), 3 out of 12 OncoKB annotated Indels and CNV showed opposite level of variant allele frequencies/CN when comparing a model derived from patient material to one developed from a PDX-derived organoid, possibly driven by a lineage-specific subclonal outgrowth when compared to patient and PDX data. Phenotypic characteristics of matched PDCs also overall show no major differences, though variability in growth rates and the ability to form spheroids in serum-free medium were noted. In one pair (919269), the PDC derived from patient material was able to form a cell line xenograft (CLX) in NSG mice but not the PDC developed from a patient-derived organoid. Overall, these models demonstrate a high degree of concordance at the genetic and phenotypic level when compared to the originating patient and/or PDX tumor. Though further characterization (e.g., preclinical drug testing) may be needed to define differences between matched PDC pairs, lack of access to patient tissue or failure to generate tumor cell cultures from one source of material should not hamper development of preclinical in vitro models from other patient-derived model types as long as the source-of-origin is clearly defined. Funded by NCI Contract No. HHSN261200800001E Citation Format: Cindy R. Timme, Ting-Chia Chang, Sergio Y. Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Alice Chen, Li Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Shahanawaz Jiwani, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Tiffanie Chase, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Yvonne A. Evrard, Mickey Williams, Dianne L. Newton, Melinda G. Hollingshead, James H. Doroshow. Comparing twenty-two matched patient-derived cell lines developed from either patient, PDX, or organoid tumor cell material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 36.

Abstract 166: Comparative analysis of response to treatments and molecular features of patient-derived tumor organoids versus cell lines and PDX derived from the same ovarian clear cell carcinoma

Abstract Preclinical models of cancer are essential for understanding cancer biology and developing effective cancer therapeutics, and potentially to guide clinical decision-making in the era of personalized medicine. Therefore, it is required that these preclinical cancer models should closely recapitulate original tumors, including histo- and molecular pathology, and match patient’s drug response. Here we described the establishment and the characterization of 7 models (4 cell lines, 2 PDTO and 1 PDX), all derived from an Ovarian Clear Cell Carcinoma (OCCC) including their responses to treatment and their comparison to the clinical response. This ovarian cancer subtype is considered as a rare tumor, clinically aggressive and chemoresistant, and very few models are yet available. We succeeded in establishing Patient-Derived Xenografts (PDX) from subcutaneous tumor fragment implantation onto nude mice, and a monolayer tumor-derived cell line and Patient-Derived Tumor Organoid (PDTO) after patient tumor dissociation. A PDTO model was derived from the PDX and 3 additional cell lines were established from this PDTO model, from the PDTO derived from the patient tumor and from the PDX model, respectively. In order to determine the relevance of each of these models, comprehensive characterization was performed based on morphological, histological, and transcriptomic analyses as well as on the evaluation of their response to the conventional treatments received by the patient. These results were compared to the features of the original tumor and to the clinical data when it was possible. Only the PDX and PDTO model derived from the patient tumor were able to retain the two components within the patient tumor; one component displaying histological features of OCCC and the other one consisted of undifferentiated cells. The patient from which all these models were derived was refractory to carboplatin as first line chemotherapy and resistant to the next lines of treatments, doxorubicin and gemcitabine, respectively. While tumor cell lines were sensitive to these treatments, PDX and PDTO models displayed resistance to the 3 drugs. The transcriptomic analysis was also consistent with these results since the models recapitulating faithfully the clinical response did cluster together away from the other classical cell culture models. Then we investigated drugs that were not used in the patient clinical management and we identified the HDAC inhibitor Belinostat as a potential effective treatments based on the response of PDTO. In conclusion, PDX and PDTO appear thus to be the most relevant models, but PDTO seem to present all the necessary prerequisites for predictive purposes and could constitute relevant tools for therapeutic decision support in the context of these particularly aggressive cancers refractory to conventional treatments. Citation Format: Lucie Thorel, Pierre-Marie Morice, Hippolyte Paysant, Romane Florent, Guillaume Babin, Cecilia Thomine, Marion Perreard, Edwige Abeilard, Florence Giffard, Emilie Brotin, Christophe Denoyelle, Celine Villenet, Melanie Briand, Alexandra Leconte, Florence Joly, Enora Dolivet, Didier Goux, Cecile Blanc-Fournier, Corinne Jeanne, Marie Villedieu, Matthieu Meryet-Figuiere, Martin Figeac, Laurent Poulain, Louis-Bastien Weiswald. Comparative analysis of response to treatments and molecular features of patient-derived tumor organoids versus cell lines and PDX derived from the same ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 166.

Abstract 1612: Therapeutic co-targeting YAP1 and TAZ using antisense oligos (ASOs) suppress gastric cancer progression and peritoneal metastases

Abstract Gastric adenocarcinoma (GAC) is one of the most common malignancies worldwide and it is the fourth leading cause of cancer-related death. Peritoneal carcinomatosis (PC; malignant ascites or implants) in GAC patients is common and poses a challenge with short survival and lack of effective therapeutics. We and others have demonstrated that deregulation of the Hippo signaling pathway particularly with upregulation of its coactivators-YAP1 or TAZ drive cancer progression and metastases in gastroesophageal cancers suggesting YAP1 and TAZ are potential drug targets in solid tumors. However, discovery of effective drugs to target YAP1 or TAZ remains challenging due to the nuclear localization and lack of inhibitory pocket for YAP1 or TAZ. In this study, using scRNAseq and immunofluorescent staining, we observed that both YAP1, TAZ and their transcriptional factors-Tead1, Tead2, Tead3 and Tead4 are highly expressed in PC tumor cells and high expression of these proteins were associated with poorer prognosis. Further, we note that recently developed YAP or TAZ antisense oligonucleotides (ASO) can effectively and specifically suppress YAP or TAZ expression and transcription accompanied by decreased tumor cell invasion and tumor sphere formation. Further, we observed that YAP1 can interact with TAZ and both bind TEAD1,2,3 but not Tead4 transcriptional factors, while YAP1 ASO or TAZ ASO can decrease the interactions between YAP1 or TAZ with TEAD1,2,3 in GAC cells. Interestingly, inhibition of YAP1 or TAZ alone using the ASOs can complementarily increase the other at the protein and mRNA levels. Further, we revealed that YAP1 KO patient derived tumor cells (GA0518) are more sensitive to TAZ ASO than control cells and simultaneously inhibition of YAP1 and TAZ by ASOs reduces both YAP1and TAZ proteins and mRNA levels with significant decrease in cell proliferation and invasive capacity of YAP1 high tumor cells. Most importantly, co-targeting YAP and TAZ by the ASOs significantly attenuated progression and PC in the PDX model and sensitized to anti-PD1 immunotherapy in the KP-Luc syngeneic model. Taken together, our studies open a new avenue for developing novel therapeutic strategy by co-targeting both YAP1 and TAZ using the ASOs against GAC with PC. Keywords: Gastric Adenocarcinoma, YAP1, TAZ, Hippo pathway, Antisense Oligonucleotides (ASOs), Peritoneal Metastases, Targeted Therapy Citation Format: Jingjing Wu, Ailing Scott, Yan Xu, Yuan Li, Yibo Fan, Ruiping Wang, Xiaodan Yao, Katsuhiro Yoshimura, Melissa Pool Pizzi, Kohei Yamashita, Shan Shao, Christopher Vellano, Linghua Wang, Alexey Revenko, Eric Dolinski, Jaffer A. Ajani, Shumei Song. Therapeutic co-targeting YAP1 and TAZ using antisense oligos (ASOs) suppress gastric cancer progression and peritoneal metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1612.

Abstract 160: Derivation and long-term maintenance of patient-derived tumoroid lines in a defined, serum-free medium

Abstract In vitro cancer research often fails to translate to the clinic, in part due to the use of traditional 2D cancer cell lines as models, which fail to resemble primary cancer cells by a variety of measures, including high mutational burden. An emerging solution to this problem is to replace traditional cell lines with patient tissue-derived cells expanded in 3D, also known as tumoroids or cancer organoids. We developed a defined, serum- and conditioned medium-free system, GibcoTM OncoProTM Tumoroid Culture Medium, that can be used to derive stable tumoroid lines from a variety of tissue sources and maintains the phenotype and genotype of patient-derived tumor cells. By supplementing the base medium with indication-specific growth factors, tumoroid lines were derived from colorectal, lung, and endometrial cancers from both fresh surgical resections and cryopreserved primary cancer cells. To demonstrate the utility of these patient-derived cells as long-term in vitro models, colorectal and lung tumoroid lines were derived from multiple donors and cultured for up to 50 passages. Brightfield microscopy, cell counts, and next-generation sequencing were used to assess maintenance of tumoroid morphology, growth rate, gene expression patterns, and genomic mutations. Patient-derived tumoroid cultures adopted donor-specific morphologies that were maintained during long-term culture. Cell doubling time tended to stabilize within the first few passages as cultures established, was donor-dependent, and averaged around 65 hours for colorectal tumoroids - on par with that of traditional 2D cancer cell lines - and 90-100 hours for lung and endometrial tumoroids, respectively. Importantly, tumoroid lines maintained their gene expression pattern for over 20,000 human RefSeq genes during long-term culture, with correlation between initial tumor material and late-passage samples of R>0.8. Distinct molecular subtypes of colorectal cancer were preserved in cultured tumoroids. The allelic frequency of single nucleotide variations (SNVs) in 161 highly relevant cancer genes was also highly correlated (R>0.9) between uncultured tissue and late-passage tumoroids. Within SNVs, transition/transversion mutation ratios were conserved. Tumoroids were cryopreserved and recovered during this study, demonstrating that biobanking of colorectal and lung tumoroids should not impact their long-term stability. Finally, a subset of the derived colorectal and lung tumoroids were tested and shown to be tumorigenic in mice, where subsequent histology of the tumor was similar to that of in vitro cultures. Altogether, tumoroid derivation and culture in this novel medium enables the long-term preservation of patient-specific cellular genotype and phenotype, which should allow for expansion, biobanking, and performance of experimental repeats within the same patient tissue-derived cultures across labs and over time. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Mark Kennedy, Matt Dallas, David Kuninger. Derivation and long-term maintenance of patient-derived tumoroid lines in a defined, serum-free medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 160.

Abstract 40: NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline

Abstract The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a national repository of Patient-Derived Models (PDMs) comprised of patient-derived xenografts (PDXs), in vitro patient-derived tumor cell cultures (PDCs) and cancer associated fibroblasts (CAFs) as well as patient-derived organoids (PDOrg). These PDMs are clinically annotated with molecular information available in an easily accessible database for the extramural community. A key effort in developing these models is to develop matched models sets allowing for larger scale screening efforts using 2D or 3D models to prioritize selection of PDX models for preclinical translational research. To date, over 220 model sets with a PDX, PDOrg, and PDC from a single patient have been developed; 40 of these have matched CAF models allowing for exploration of research questions focused on tumor microenvironment. The largest model sets are in colorectal cancer (COADREAD, n=76), gynecologic cancers (n=33), pancreatic adenocarcinoma (PAAD, n=29), melanoma (MEL, n=19), and head and neck squamous cell carcinomas (HNSCC, n=17). Every model undergoes several quality control assessments that serve as go/no-go criteria including pathology assessment, STR validation, NGS concordance assessment and for PDXs, human:mouse DNA content assessment. It should be noted that not every model is successful in the development or QC phase so additional model sets with only one or two of the model types are also available for researcher requests, for example there are over 125 PDX/PDOrg matched model sets. The NCI is currently performing parallel preclinical screening of PDXs and PDCs or PDOrgs to determine the ability of the in vitro lines to predict in vivo activity. Genetic and histopathologic assessment of these matched model sets have demonstrated a high degree of stability by somatic mutation, copy number alteration (CNA) and gene expression data. Gene expression correlation analysis shows that mean of Spearman r between PDXs 0.89, between matched PDC/PDXs 0.79, and between matched PDOrg/PDXs 0.82. As expected, some variation at the gene expression level when comparing PDX to in vitro cultures by t-SNE can be observed, likely due to the differences in culture conditions. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Li Chen, Sergio Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Ting-Chia Chang, Alice Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Ahalt-Gottholm, Tara Grinnage-Pulley, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Shahanawaz Jiwani, Tiffanie Miner, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Cindy R. Timme, Dianne L. Newton, Paul Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 40.

PD17-01 THE IMPACT OF LOSS AND MUTATION OF MITOCHONDRIAL DNA ON ONCOMETABOLITE ACCUMULATION IN PATIENT-DERIVED FUMARATE HYDRATASE-DEFICIENT RENAL TUMOR CELL LINES

You have accessJournal of UrologyCME1 Apr 2023PD17-01 THE IMPACT OF LOSS AND MUTATION OF MITOCHONDRIAL DNA ON ONCOMETABOLITE ACCUMULATION IN PATIENT-DERIVED FUMARATE HYDRATASE-DEFICIENT RENAL TUMOR CELL LINES Bhargav Arimilli, Daniel Crooks, Ye Yang, Youfeng Yang, Cathy Vocke, Christopher Ricketts, Mark Ball, Teresa Fan, Andrew Lane, and W. Marston Linehan Bhargav ArimilliBhargav Arimilli More articles by this author , Daniel CrooksDaniel Crooks More articles by this author , Ye YangYe Yang More articles by this author , Youfeng YangYoufeng Yang More articles by this author , Cathy VockeCathy Vocke More articles by this author , Christopher RickettsChristopher Ricketts More articles by this author , Mark BallMark Ball More articles by this author , Teresa FanTeresa Fan More articles by this author , Andrew LaneAndrew Lane More articles by this author , and W. Marston LinehanW. Marston Linehan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant cancer syndrome characterized by mutations in the fumarate hydratase gene (FH). We recently reported the discovery of mutations and loss of mitochondrial DNA (mtDNA) in FH-deficient renal tumors and tumor-derived cell lines. Mitochondrial alterations in FH-deficient tumor-derived cell lines ranged from inactivating mutations in mitochondrial Complex I and Complex III subunits to complete loss of mtDNA in two tumor cell lines. The goal of the present study was to characterize the impact of mtDNA mutations on the oncometabolites fumarate and succinate in FH-deficient renal tumor cell lines. METHODS: We performed stable isotope-resolved metabolomics (SIRM) with 13C5,15N2-glutamine tracer on five FH-deficient patient-derived tumor cell lines, and utilized ion chromatography coupled with ultra-high resolution Orbitrap-based mass spectrometry (UHR-ICMS) and NMR spectroscopy to determine the abundance and isotopic labeling pattern of fumarate and its precursors in polar extracts obtained from the tumor cells. RESULTS: We found that several of the FH-deficient tumor cell lines showed elevated succinate levels, with a 13C labeling pattern that was consistent with oxidative production of succinate from glutamine. In contrast, fumarate 13C labeling patterns in these same cell lines was consistent with reductive carboxylation of glutamine and formation of fumarate from the urea cycle and purine biosynthesis, suggesting a deficiency of succinate dehydrogenase (Complex II) activity in the cells. UHR-ICMS analysis of FH-/- cells labeled with 13C1-glutamine confirmed 13C labeling of the purine biosynthetic intermediate adenylosuccinate via reductive carboxylation of glutamine, giving rise to 13C1-fumarate, and this 13C label was localized to the carboxylate carbon of fumarate using an HMBC NMR experiment. CONCLUSIONS: We have uncovered additional lesions in the TCA cycle in patient-derived HLRCC cell lines, giving rise to accumulation of the oncometabolite succinate. These data highlight the metabolic heterogeneity that is observed in genetically-defined human tumors. Source of Funding: NIH NIH MRSP © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e495 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bhargav Arimilli More articles by this author Daniel Crooks More articles by this author Ye Yang More articles by this author Youfeng Yang More articles by this author Cathy Vocke More articles by this author Christopher Ricketts More articles by this author Mark Ball More articles by this author Teresa Fan More articles by this author Andrew Lane More articles by this author W. Marston Linehan More articles by this author Expand All Advertisement PDF downloadLoading ...

Endemic Burkitt lymphoma avatar mouse models for exploring inter-patient tumor variation and testing targeted therapies.

Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children.

Homotypic Membrane-Enhanced Blood-Brain Barrier Crossing and Glioblastoma Targeting for Precise Surgical Resection and Photothermal Therapy.

The crossing of blood-brain barrier (BBB) is essential for glioblastoma (GBM) therapy, and homotypic targeting is an effective strategy to achieve BBB crossing. In this work, GBM patient-derived tumor cell membrane (GBM-PDTCM) is prepared to cloak gold nanorods (AuNRs). Relying on the high homology of the GBM-PDTCM to the brain cell membrane, GBM-PDTCM@AuNRs realize efficient BBB crossing and selective GBM targeting. Meanwhile, owing to the functionalization of Raman reporter and lipophilic fluorophore, GBM-PDTCM@AuNRs are able to generate fluorescence and Raman signals at GBM lesion, and almost all tumor can be precisely resected in 15 min by the guidance of dual signals, ameliorating the surgical treatment for advanced GBM. In addition, photothermal therapy for orthotopic xenograft mice is accomplished by intravenous injection of GBM-PDTCM@AuNRs, doubling the median survival time of the mice, which improves the nonsurgical treatment for early GBM. Therefore, benefiting from homotypic membrane-enhanced BBB crossing and GBM targeting, all-stage GBM can be treated with GBM-PDTCM@AuNRs in distinct ways, providing an alternative idea for the therapy of tumor in the brain.

YAP1 and WWTR1 expression inversely correlates with neuroendocrine markers in Merkel cell carcinoma.

BackgroundMerkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable, and the expression profile of MCC tumors is, to our knowledge, unexplored.MethodsHere, we leveraged bulk and single-cell RNA-Seq of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional environment of MCC.ResultsStrikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain-containing transcriptional regulator 1 (WWTR1). This inverse correlation was broadly present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEA domain-dependent (TEAD-dependent) transcriptional repression of MCPyV LT.ConclusionThese findings identify what we believe to be a previously unrecognized heterogeneity in NE gene expression within MCC and support a model of YAP1/WWTR1 silencing as essential for the development of MCPyV-positive MCC.FundingUS Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.

Do we need more clinical trials?

Do we need more clinical trials?

Modeling interaction of Glioma cells and CAR T-cells considering multiple CAR T-cells bindings

Chimeric antigen receptor (CAR) T-cell based immunotherapy has shown its potential in treating blood cancers, and its application to solid tumors is currently being extensively investigated. For glioma brain tumors, various CAR T-cell targets include IL13Rα2, EGFRvIII, HER2, EphA2, GD2, B7-H3, and chlorotoxin. In this work, we are interested in developing a mathematical model of IL13Rα2 targeting CAR T-cells for treating glioma. We focus on extending the work of Kuznetsov et al. (1994) by considering binding of multiple CAR T-cells to a single glioma cell, and the dynamics of these multi-cellular conjugates. Our model more accurately describes experimentally observed CAR T-cell killing assay data than the models which do not consider multi-cellular conjugates. Moreover, we derive conditions in the CAR T-cell expansion rate that determines treatment success or failure. Finally, we show that our model captures distinct CAR T-cell killing dynamics from low to high antigen receptor densities in patient-derived brain tumor cells.