Patient-derived Tumor Models Research Articles

Abstract LB004: Upregulation of STAT3 signaling in tumor cells fosters a TME-dependent secondary resistance of BRCA1-deficient HGSOC to PARP inhibition

Abstract PARP inhibitors (PARPi) have demonstrated potent therapeutic efficacy in patients with ovarian cancer. However, acquired resistance to PARPi is a major challenge in the clinic. Therefore, understanding the resistance mechanism and developing new treatment approaches are important to overcome therapeutic resistance to PARP inhibition. By using our GEM of Brca1-deficient ovarian tumor model, we uncovered a new mechanism underlying a secondary resistance to PARP inhibition mediated by tumor associated macrophages (TAMs) in the tumor microenvironment (TME). Mechanistically, PARP inhibition induced the STAT3 signaling pathway in tumor cells, which in turn promoted pro-tumor polarization of TAMs in the TME of ovarian cancer. Ablation of STAT3 in tumor cells mitigated polarization of M2-like pro-tumor macrophages in the TME and increased tumor infiltration T cells in response to PARP inhibition. Furthermore, we demonstrated that STING agonists reprogramed myeloid cells in the TME of ovarian tumor into antitumor M1-like macrophages and activated dendritic cells (DCs) in a myeloid cell STING-dependent manner. These findings were recapitulated in patient-derived PARPi-resistant ovarian tumor models. Finally, we show that STING agonism was able to overcome the secondary resistance to PARPi in ovarian cancer rendered by immunosuppressive TME. Our study elucidates a new mechanism of PARPi resistance and provides a new treatment strategy to overcome the TME-induced therapeutic resistance to PARP inhibition in ovarian cancers. Citation Format: Liya Ding, Qiwei Wang, Michael Kearns, Tao Jiang, Xin Cheng, Changli Qian, Jean Zhao. Upregulation of STAT3 signaling in tumor cells fosters a TME-dependent secondary resistance of BRCA1-deficient HGSOC to PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB004.

Abstract 3332: LNG-451 is a potent, CNS-penetrant, wild-type EGFR sparing inhibitor of EGFR exon 20 insertion mutations

Abstract Exon 20 insertion mutations (Ex20ins) are the third most common type of EGFR oncogenic mutation in lung cancer patients. While several small molecules have been approved (mobocertinib) or are in clinical development (e.g. CLN-081, BDTX-189) none have demonstrated meaningful CNS activity and they can be associated with treatment-limiting adverse events, including wild-type (WT) EGFR-mediated toxicities. LNG-451 is a CNS-penetrant, wild-type EGFR-sparing, covalent inhibitor of EGFR Ex20ins. In cell viability assays dependent on five EGFR Ex20ins mutations, LNG-451 was a potent inhibitor (IC50 7 - 78 nM) including the three most prevalent insertions (V769_D770insASV IC50 = 78 nM, D770_N771insSVD IC50 = 53 nM, H773_V774insNPH IC50 = 75 nM). LNG-451 had low potency in WT EGFR dependent cell assays (WT EGFR Ba/F3 IC50=1,960 nM, cell panel IC50 = 1630 nM). LNG-451 was 21-fold selective for its least potent Ex20ins (V769_D770insASV) relative to WT EGFR. LNG-451 potently inhibited cells dependent on EGFR L861Q (IC50 = 6 nM), EGFR G719S (IC50 = 8 nM), and EGFR G719S/T263P (IC50 = 11 nM). LNG-451 is a highly selective kinase inhibitor (1 μM LNG-451, >90% inhibition for 7 of 409 WT kinases) with screening hits having a similarly positioned cysteine as EGFR. In a Ba/F3 cell-derived xenograft model harboring an EGFR V769_D770insASV exon 20 insertion mutation (EGFR V769_D770insASV CDX), 10 and 50 mg/kg LNG-451 QD PO dosing for 15 days was well-tolerated and resulted in tumor growth inhibition (TGI) of 75.7% and 105.4%, respectively. LNG-451 was efficacious and well-tolerated in the EGFR Ex20ins HuPrime® LU0387 H773-V774insNPH patient-derived xenograft tumor model: 10 mg/kg LNG-451 QD, 67% TGI; 50 mg/kg LNG-451 QD, 91% tumor regression. LNG-451 was highly active in the intracranial PC9-luc xenograft mouse model. In a single dose PK/PD study using the EGFR V769_D770insASV CDX, similar LNG-451 concentrations were observed in tumor, large intestine, and skin tissues (e.g. 50 mg/kg LNG-451 3h post dose: tumor, 8387 ng/mL; large intestine tissue, 14483 ng/mL; skin, 4623 ng/mL). LNG-451 potently suppressed EGFR phosphorylation in tumor tissue (e.g. 99%, 3 h post 50 mg/kg dose) but had minimal-to modest suppression of phospho-EGFR in large intestine tissue (e.g. 4.2%, 3h post 50 mg/kg dose) and skin tissue (e.g 1.9%, 3h post 50 mg/kg dose). Taken together, LNG-451 is a wild-type EGFR-sparing, CNS-penetrant EGFR Ex20ins inhibitor that is expected to provide strong anti-tumor efficacy for patients with advanced/metastatic solid cancers harboring oncogenic EGFR exon 20 insertions with reduced WT EGFR-driven toxicities. Citation Format: Brion W. Murray, Anjali Pandey, Bruce Roth, Tracy Saxton, Daniel J. Estes, Himanshu Agrawal, Santosh Vishwakarma, Gurulingappa Hallur, Ishtiyaque Ahmad, Ravi Trivedi, Helen Jenkins, Paul G. Pearson. LNG-451 is a potent, CNS-penetrant, wild-type EGFR sparing inhibitor of EGFR exon 20 insertion mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3332.

Abstract 5363: The preclinical selectivity and activity of APS03118, a highly selective and potent next-generation RET inhibitor

Abstract Backgroud: Oncogenic RET is an actionable target across a variety of cancers. Selective RET inhibitors selpercatinib and pralsetinib were recently approved by the FDA and EMA for patients with RET-dependent NSCLC and thyroid cancers. The solvent front mutations (SFMs) RET G810C/S/R have been identified as mechanisms of acquired resistance to both drugs. The gatekeeper RET V804 mutation cannot be inhibited by selected next-generation RET inhibitors in development. APS03118 is a novel next-generation RET inhibitor which is potent against a range of RET fusions and mutations including both SFMs and gatekeeper mutations. Methods: The selectivity, anti-RET activity, and intracranial efficacy of APS03118 were confirmed in vitro and in vivo in a variety of RET-dependent tumor models. Results: APS03118 was highly selective against a panel of 468 kinases and demonstrated 130-fold selectivity over VEGFR-2. In enzymatic assays, APS03118 showed low nanomolar potency against wild type RET and 25 RET mutations/fusions, including the inhibition of RET G810R/C/S (IC50 0.04-5 nM) and RET V804M/L/E (IC50 0.04-1 nM). APS03118 inhibited RET phosphorylation (IC50 <15 nM) in Ba/F3 engineered RET cells (WT, G810R, V804M, M918T). In cell proliferation assays, APS03118 potently inhibited KIF5B-RET Ba/F3 (WT, V804M, V804L, M918T), CCDC6-RET Ba/F3 (WT, V804M, S904F), LC2/ad (CCDC6-RET), TT (RET C634W) (IC50 < 10nM); Ba/F3 RET G810R and G810S IC50 (8-65 nM). APS03118 demonstrated marked anti-tumor efficacy in vivo in RET-driven cell-derived (Ba/F3 KIF5B-RET, V804M, TT (C634W)) and patient-derived (KIF5B-RET, CCDC6-RET, CCDC6-RET V804M) xenograft tumor models. At 10 mg/kg BID, tumor regression was observed in these xenograft models (TGI 87-108%). Tumors completely subsided in CCDC6-RET orthotopic brain model with a 100% survival rate. In the Ba/F3 KIF5B-RET G810R xenograft model, APS03118 30 mg/kg BID showed 90% TGI and was well tolerated, and RET G810 mutations often drive clinical progression on current RET inhibitors. Conclusions: APS03118 is a novel highly selective next-generation RET inhibitor that possesses potent in vitro and in vivo activity against a diverse range of RET alterations, including SFMs-mediated resistance. A first-in-human phase 1 trial for patients with RET-driven solid tumors with activating RET alterations is planned for 2022. Citation Format: Alexander Drilon, Jun Zhong, Ying Lu, Yongbo Liu, Hao Wang, Minchun Chen, Xiaohu Chen, John Zhu, Shun Lu, Vivek Subbiah. The preclinical selectivity and activity of APS03118, a highly selective and potent next-generation RET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5363.

Abstract 6330: NKT2152: A highly potent HIF2α inhibitor and its therapeutic potential in solid tumors beyond ccRCC

Abstract Clear cell Renal Cell Carcinoma (ccRCC) is characterized in most cases (>80%) by the inactivation of von Hippel-Lindau (VHL) tumor suppressor. VHL loss leads to HIF2α accumulation and constitutive activation of its downstream genes important in carcinogenesis. Preclinical and clinical evidence demonstrate that HIF2α inhibition by the first-in-class belzutifan (MK-6482) offers an effective treatment for ccRCC. Here we discovered a novel, potent, selective, and orally available small molecule HIF2α inhibitor (NKT2152) through rational drug design. Cellular thermal shift assay demonstrated NKT2152 engaged HIF2α and stabilized it against heat-induced denaturation in ccRCC 786-O cells. NKT2152 disrupted HIF2α/HIF1β complex in a dose-dependent manner, resulting in increased cytosol localization and reduced nucleus localization of HIF2α in 786-O cells. In vitro, NKT2152 specifically suppressed the expression of HIF2α target genes including vascular endothelial growth factor A, cyclin D1, and glucose transporter 1 in 786-O cells. Pharmacodynamic analysis of NKT2152 in the 786-O xenograft tumor model confirmed that NKT2152 potently inhibited HIF2α as demonstrated by the repression of HIF2α-regulated genes at both mRNA and protein levels. Moreover, NKT2152 treatment, administrated twice daily by oral gavage, led to a dose-dependent tumor growth inhibition or tumor regression in both ccRCC cell line-derived (A498 and 786-O) and patient-derived xenograft tumor models. More pronounced tumor growth inhibition was observed when NKT2152 was combined with a VEGFR inhibitor or a CDK4/6 inhibitor in the 786-O xenograft tumor model. In addition to directly promoting tumor progression by regulating gene expression in tumor cells, HIF2α has also been proposed to play an integral role in shaping the tumor microenvironment under hypoxia. Given hypoxia is a hallmark of most solid tumors and is associated with poor survival in a wide range of cancer types, we tested the hypothesis that HIF2α inhibition by NKT2152 may exert broader anti-tumor activity in other solid tumors that lack a VHL gene deficiency. Indeed, we demonstrated here that NKT2152 caused significant tumor growth inhibition in multiple solid tumor xenograft models, including but not limited to hepatocellular carcinoma. NKT2152 had excellent oral bioavailability and achieved high exposures in mouse, rat and dog. These data not only support clinical development of NKT2152 in ccRCC, but also provide exciting opportunities for expanding to other solid tumor indications with tremendous unmet medical needs. Clinical evaluation of NKT2152 for the treatment of ccRCC is ongoing. Citation Format: Jing Lu, Hairong Wei, Wenfeng Sun, Jianlin Geng, Ke Liu, Jessica Liu, Zhihong Liu, Jiping Fu, Yigang He, Keshi Wang, Yan Lou, Zhenhai Gao. NKT2152: A highly potent HIF2α inhibitor and its therapeutic potential in solid tumors beyond ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6330.

Abstract 3831: Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms

Abstract Triple-negative breast cancer (TNBC) is an aggressive, heterogenous disease that lacks approved targeted therapeutics. Due to the complexity of tumor microenvironment (TME) interactions in TNBC, it is crucial to identify therapeutic targets that regulate the extracellular matrix (ECM). Traditional 2D TNBC models do not accurately mimic the cell-matrix interactions and structural matrix proteins that are key in maintaining the unique TME in individual patient tumors. Here, we introduce our decellularized TNBC patient-derived xenograft (PDX) models to address this gap in knowledge. Utilizing our decellularized models, we can investigate the unique ECM composition and cell-matrix interactions in PDX models from understudied patients with diverse clinical presentations. We can also use our decellularized tumor scaffolds as drug testing models. We utilize our tissue decellularization method on PDX tumor scaffolds derived from two of our established TNBC PDX models (TU-BcX-56S and TU-BcX-4QX). Decellularization of tumor scaffolds was confirmed by DNA quantification and histological analysis (H&E, Masson’s Trichrome, and Movat’s staining). MDA-MB-231 cells expressing GFP and luciferase were seeded onto scaffolds and treated with 100nM of Paclitaxel, a microtubule-stabilizing cytotoxic chemotherapeutic, or DMSO for 48 hours. Concordantly, cells in 2D culture were treated with 100nM of Paclitaxel or DMSO for 48 hours. Using the IVIS imaging system, we measured the bioluminescence of cells on our seeded scaffolds before treatment and at 24 and 48 hours after treatment. Cells in 2D culture showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Cells seeded on TU-BcX-56S scaffold also showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Interestingly, cells seeded on TU-BcX-4QX scaffold showed a decrease in bioluminescence in both the treatment and control groups at 24 and 48 hours, albeit there was no significant difference. Using qPCR, we investigated scaffold regulation of ECM gene composition in our treatment and control groups. These results demonstrate that the unique ECM composition and architecture of these scaffolds are important drivers of cell behavior. Here, we demonstrate that our decellularized PDX tumor scaffold models can be used as drug testing platforms. Furthermore, our models recapitulate key structural matrix proteins in the TNBC TME, can be used to investigate ECM onco-architecture and composition, and can be used in therapeutic discovery research. Citation Format: Maryl Wright, Khoa Nguyen, Elizabeth Martin, Melyssa Bratton, Bridgette Collins-Burow, Matthew Burow. Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3831.

A Platform of Patient-Derived Microtumors Identifies Individual Treatment Responses and Therapeutic Vulnerabilities in Ovarian Cancer.

Simple SummaryFor personalized oncology, it is crucial to develop appropriate patient-derived tumor models that allow individualized validation of the most effective cancer therapy. The objective of this study was to develop and characterize a new patient-derived ovarian cancer tumor model composed of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TIL). In contrast to other preclinical tumor models, such as patient-derived organoids, PDM are generated within 24 h from fresh ovarian tumor samples. From immunohistochemical comparison with the original primary tumor, we conclude that the histopathological features of the original tumor are essentially preserved. Importantly, we successfully identified treatment-sensitive and treatment-resistant tumor models for standard platinum-based therapy by reverse-phase protein array (RPPA) analysis of PDM. Furthermore, we were able to evaluate the efficacy of cancer immunotherapy by co-culturing PDM and autologous TILs. PDM and TILs may therefore serve as a preclinical platform to identify individualized, tailored cancer treatments in the future.In light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease, we developed a 3D preclinical tumor model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) to identify individual treatment vulnerabilities and validate chemo-, immuno- and targeted therapy efficacies. Enzymatic digestion of primary ovarian cancer tissue and cultivation in defined serum-free media allowed rapid and efficient recovery of PDM, while preserving histopathological features of corresponding patient tumor tissue. Reverse-phase protein array (RPPA)-analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Co-cultures of PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Combining protein pathway analysis and drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision making.

Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer.

Telomere maintenance is a hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying clinically translatable imaging biomarkers of TERT can enable noninvasive assessment of tumor proliferation and response to therapy. We used RNAi, doxycycline-inducible expression systems, and pharmacologic inhibitors to mechanistically delineate the association between TERT and metabolism in preclinical patient-derived tumor models. Deuterium magnetic resonance spectroscopy (2H-MRS), which is a novel, translational metabolic imaging modality, was used for imaging TERT in cells and tumor-bearing mice in vivo. Our results indicate that TERT expression is associated with elevated NADH in multiple cancers, including glioblastoma, oligodendroglioma, melanoma, neuroblastoma, and hepatocellular carcinoma. Mechanistically, TERT acts via the metabolic regulator FOXO1 to upregulate nicotinamide phosphoribosyl transferase, which is the key enzyme for NAD+ biosynthesis, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. Because NADH is essential for pyruvate flux to lactate, we show that 2H-MRS-based assessment of lactate production from [U-2H]-pyruvate reports on TERT expression in preclinical tumor models in vivo, including at clinical field strength (3T). Importantly, [U-2H]-pyruvate reports on early response to therapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by MRI. Elevated NADH is a metabolic consequence of TERT expression in cancer. Importantly, [U-2H]-pyruvate reports on early response to therapy, prior to anatomic alterations, thereby providing clinicians with a novel tool for assessment of tumor burden and treatment response in cancer.

The development of APS03118, a potent next-generation RET inhibitor for treating RET-inhibitor-resistant patients.

e15107 Background: Oncogenic RET is an actionable target across a variety of cancers. Selective RET inhibitors selpercatinib and pralsetinib were recently approved by the FDA and EMA for patients with RET-dependent NSCLC and thyroid cancers. The solvent front mutations (SFMs) RET G810C/S/R have been identified as mechanisms of acquired resistance to both drugs. APS03118 is a novel next-generation RET inhibitor which is potent against a range of RET fusions and mutations including both SFMs and gatekeeper mutations. Methods: The selectivity, anti-RET activity, and intracranial efficacy of APS03118 were confirmed in vitro and in vivo in a variety of RET-dependent tumor models. Results: APS03118 was highly selective against a panel of 468 kinases and demonstrated 130-fold selectivity over VEGFR-2. In enzymatic assays, APS03118 showed low nanomolar potency against wild type RET and 25 RET mutations/fusions, including the inhibition of RET G810R/C/S (IC50 0.04-5 nM) and RET V804M/L/E (IC50 0.04-1 nM). APS03118 inhibited RET phosphorylation (IC50 < 15 nM) in Ba/F3 engineered RET cells (WT, G810R, V804M, M918T). In cell proliferation assays, APS03118 potently inhibited the proliferation of KIF5B-RET Ba/F3 (WT, V804M, V804L, M918T), CCDC6-RET Ba/F3 (WT, V804M, S904F), LC2/ad (CCDC6-RET), TT (RET C634W) (IC50 < 10nM); Ba/F3 RET G810R and G810S IC50 (8-65 nM). APS03118 demonstrated marked anti-tumor efficacy in vivo in RET-driven cell-derived (Ba/F3 KIF5B-RET, V804M, TT (C634W)) and patient-derived (KIF5B-RET, CCDC6-RET, CCDC6-RET V804M) xenograft tumor models at 10 mg/kg (TGI 87-108%). Tumors completely subsided in CCDC6-RET orthotopic brain model with a 100% survival rate. In the Ba/F3 KIF5B-RET G810R xenograft model, APS03118 30 mg/kg showed 90% TGI and was well tolerated, and RET G810 mutations often drive clinical progression on current RET inhibitors. The pharmacokinetic/pharmacodynamic relationship of APS03118 in Ba/F3 KIF5B-RET G810R and WT xenograft tumor model was investigated, and the sustained decrease of the phosphorylated RET were observed at 30 mg/kg with the plasma exposure exceed cell IC90. Conclusions: APS03118 is a novel highly selective next-generation RET inhibitor that possesses potent in vitro and in vivo activity against a diverse range of RET alterations, including SFMs-mediated resistance. APS03118 has received IND approval and Fast Track Designation from FDA, and a first-in-human phase 1 trial for patients with RET-driven solid tumors with activating RET alterations is planned for 2022.

Combinatorial targeting of Hippo-STRIPAK and PARP elicits synthetic lethality in gastrointestinal cancers.

The striatin-interacting phosphatase and kinase (STRIPAK) complexes integrate extracellular stimuli that result in intracellular activities. Previously, we discovered that STRIPAK is a key machinery responsible for loss of the Hippo tumor suppressor signal in cancer. Here, we identified the Hippo-STRIPAK complex as an essential player in the control of DNA double-stranded break (DSB) repair and genomic stability. Specifically, we found that the mammalian STE20-like protein kinases 1 and 2 (MST1/2), independent of classical Hippo signaling, directly phosphorylated zinc finger MYND type–containing 8 (ZMYND8) and hence resulted in the suppression of DNA repair in the nucleus. In response to genotoxic stress, the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway was determined to relay nuclear DNA damage signals to the dynamic assembly of Hippo-STRIPAK via TANK-binding kinase 1–induced (TBK1-induced) structural stabilization of the suppressor of IKBKE 1– sarcolemma membrane–associated protein (SIKE1-SLMAP) arm. As such, we found that STRIPAK-mediated MST1/2 inactivation increased the DSB repair capacity of cancer cells and endowed these cells with resistance to radio- and chemotherapy and poly(ADP-ribose)polymerase (PARP) inhibition. Importantly, targeting the STRIPAK assembly with each of 3 distinct peptide inhibitors efficiently recovered the kinase activity of MST1/2 to suppress DNA repair and resensitize cancer cells to PARP inhibitors in both animal- and patient-derived tumor models. Overall, our findings not only uncover what we believe to be a previously unrecognized role for STRIPAK in modulating DSB repair but also provide translational implications of cotargeting STRIPAK and PARP for a new type of synthetic lethality anticancer therapy.

3D patient-derived tumor models to recapitulate pediatric brain tumors In Vitro

Brain tumors are the leading cause of cancer-related deaths in children. Tailored therapies need preclinical brain tumor models representing a wide range of molecular subtypes. Here, we adapted a previously established brain tissue-model to fresh patient tumor cells with the goal of establishing3D in vitro culture conditions for each tumor type.Wereported our findings from 11 pediatric tumor cases, consisting of three medulloblastoma (MB) patients, three ependymoma (EPN) patients, one glioblastoma (GBM) patient, and four juvenile pilocytic astrocytoma (Ast) patients. Chemically defined media consisting of a mixture of pro-neural and pro-endothelial cell culture medium was found to support better growth than serum-containing medium for all the tumor cases we tested. 3D scaffold alone was found to support cell heterogeneity and tumor type-dependent spheroid-forming ability; both properties were lost in 2D or gel-only control cultures. Limited in vitro models showed that the number of differentially expressed genes between in vitro vs. primary tissues, are 104 (0.6%) of medulloblastoma, 3,392 (20.2%) of ependymoma, and 576 (3.4%) of astrocytoma, out of total 16,795 protein-coding genes and lincRNAs. Two models derived from a same medulloblastoma patient clustered together with the patient-matched primary tumor tissue; both models were 3D scaffold-only in Neurobasal and EGM 1:1 (v/v) mixture and differed by a 1-mo gap in culture (i.e., 6wk versus 10wk). The genes underlying the in vitrovs. in vivo tissue differences may provide mechanistic insights into the tumor microenvironment. This study is the first step towards establishing a pipeline from patient cells to models to personalized drug testing for brain cancer.

Patient-derived tumor models are attractive tools to repurpose drugs for ovarian cancer treatment: pre-clinical updates.

Despite advances in understanding of ovarian cancer biology, the progress in translation of research findings into new therapies is still slow. It is associated in part with limitations of commonly used cancer models such as cell lines and genetically engineered mouse models that lack proper representation of diversity and complexity of actual human tumors. In addition, the development of de novo anticancer drugs is a lengthy and expensive process. A promising alternative to new drug development is repurposing existing FDA-approved drugs without primary oncological purpose. These approved agents have known pharmacokinetics, pharmacodynamics, and toxicology and could be approved as anticancer drugs quicker and at lower cost. To successfully translate repurposed drugs to clinical application, an intermediate step of pre-clinical animal studies is required. To address challenges associated with reliability of tumor models for pre-clinical studies, there has been an increase in development of patient-derived xenografts (PDXs), which retain key characteristics of the original patient’s tumor, including histologic, biologic, and genetic features. The expansion and utilization of clinically and molecularly annotated PDX models derived from different ovarian cancer subtypes could substantially aid development of new therapies or rapid approval of repurposed drugs to improve treatment options for ovarian cancer patients.

A Comparative Study of Patient-Derived Tumor Models of Pancreatic Ductal Adenocarcinoma Involving Orthotopic Implantation

Background: Pancreatic ductal adenocarcinoma (PDA) is associated with very poor prognoses. Therefore, new therapies and preclinical models are urgently needed. In the present study, we sought to develop more realistic experimental models for use in PDA research. Methods: We developed patient-derived xenografts (PDXs), established PDX-derived cell lines (PDCLs), and generated cell line-derived xenografts (CDXs), which we integrated to create 13 matched “trios” – i.e., patient-derived tumor models of PDA. We then compared and contrasted histological and molecular alterations between these three model systems. Results: Orthotopic implantation (OI) of the PDCLs resulted in tumorigenesis and metastases to the liver and peritoneum. Morphological comparisons of OI-CDXs and OI-PDXs with passaged tumors revealed that the histopathological features of the original tumor were maintained in both models. Molecular alterations in PDX tumors (including those to KRAS, TP53, SMAD4, and CDKN2A) were similar to those in the respective PDCLs and CDX tumors. When gene expression levels in the PDCLs, ectopic tumors, and OI tumors were compared, the distant metastasis-promoting gene CXCR4 was specifically upregulated in OI tumors, whose immunohistochemical profiles suggested epithelial-mesenchymal transition and adeno-squamous trans-differentiation. Conclusion: These patient-derived tumor models provide useful tools for monitoring responses to antineoplastic agents and for studying PDA biology.

89Zr]ZrDFO-CR011 PET Correlates with Response to Glycoprotein Nonmetastatic Melanoma B-targeted Therapy in Triple-negative Breast Cancer.

There is a need for prognostic markers to select patients most likely to benefit from antibody-drug conjugate (ADC) therapy. We quantified the relationship between pretreatment PET imaging of glycoprotein nonmetastatic melanoma B (gpNMB) with 89Zr-labeled anti-gpNMB antibody ([89Zr]ZrDFO-CR011) and response to ADC therapy (CDX-011) in triple-negative breast cancer. First, we compared different PET imaging metrics and found that standardized uptake values (SUV) and tumor-to-heart SUV ratios were sufficient to delineate differences in radiotracer uptake in the tumor of four different cell- and patient-derived tumor models and achieved high standardized effect sizes. These tumor models with varying levels of gpNMB expression were imaged with [89Zr]ZrDFO-CR011 followed by treatment with a single bolus injection of CDX-011. The percent change in tumor volume relative to baseline (% CTV) was then correlated with SUVmean of [89Zr]ZrDFO-CR011 uptake in the tumor. All gpNMB-positive tumor models responded to CDX-011 over 6 weeks of treatment, except one patient-derived tumor regrew after 4 weeks of treatment. As expected, the gpNMB-negative tumor increased in volume by 130 ± 59% at endpoint. The magnitude of pretreatment SUV had the strongest inverse correlation with the % CTV at 2-4 weeks after treatment with CDX-011 (Spearman ρ = -0.8). However, pretreatment PET imaging with [89Zr]ZrDFO-CR011 did not inform on which tumor types will regrow over time. Other methods will be needed to predict resistance to treatment.

Development of a Dendritic Cell/Tumor Cell Fusion Cell Membrane Nano-Vaccine for the Treatment of Ovarian Cancer.

Ovarian cancer (OC) is a malignant tumor that seriously affects women’s health. In recent years, immunotherapy has shown great potential in tumor treatment. As a major contributor of immunotherapy, dendritic cells (DCs) - based tumor vaccine has been demonstrated to have a positive effect in inducing immune responses in animal experiments. However, the effect of tumor vaccines in clinical trials is not ideal. Therefore, it is urgent to improve the existing tumor vaccines for tumor treatment. Here, we developed a fusion cell membrane (FCM) nano-vaccine FCM-NPs, which is prepared by fusing DCs and OC cells and coating the FCM on the poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with the immune adjuvant CpG-oligodeoxynucleotide (CpG-ODN). The fusion process promoted the maturation of DCs, thus up-regulating the expression of costimulatory molecule CD80/CD86 and accelerating lymph node homing of DCs. Furthermore, FCM-NPs has both the immunogenicity of tumor cells and the antigen presenting ability of DCs, it can stimulate naive T lymphocytes to produce a large number of tumor-specific cytotoxic CD8+ T lymphocytes. FCM-NPs exhibited strong immuno-activating effect both in vitro and in vivo. By establishing subcutaneous transplanted tumor model, patient-derived xenograft tumor model and abdominal metastatic tumor model, FCM-NPs was proved to have the effect of delaying the growth and inhibiting the metastasis of OC. FCM-NPs is expected to become a new tumor vaccine for the treatment of ovarian cancer.

Abstract P5-06-15: Enabling therapeutic decisions for a breast cancer patient cohort using matched diseased and normal tissue in tumor organoids

Abstract Pre-clinical cancer studies have been limited by the availability of patient cell models that accurately represent the diversity of patient populations. The utility of these studies is further impacted by the lack of normal tissue from the same patient. Furthermore, clinical evaluation of targeted therapeutics, like tarceva (erlotinib), would benefit from patient models expressing the target for robust characterization of drug response. Moreover, individual patient responses to the same anti-cancer compound cannot be assessed with high precision unless healthy controls are used from the same tumor donor. Here, we present six breast cancer patient-derived tumor models, accompanied by cancer (tumor)-associated fibroblasts (CAFs) and mesenchymal stem cells (MSCs). These paired and matched sample cohorts were studied in the context of spheroid models of the metastatic breast cancer niche. We show that these models allow an increased understanding of chemotherapy modulation by non-cancerous cells, which are present within the tumor but not targeted by the current chemotherapies or other anti-tumor approaches. We used a three-drug set to create a viability profile for each patient-associated tumor sample, which in turn can be used to aid clinical decision-making and increase the personalization of each treatment for an individual. Further, our approach shows different genomic and proteomic profiles for the co-culture models when compared between the tumor 3D and healthy controls. Citation Format: Liam Deems, Dmitry Shvartsman, Maria Ivanova, Cheryl Murphy, David Deems. Enabling therapeutic decisions for a breast cancer patient cohort using matched diseased and normal tissue in tumor organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-15.

Establishment of a mouse model of pancreatic cancer using human pancreatic cancer cell line S2-013-derived organoid

A well-established preclinical model of pancreatic cancer needs to be established to facilitate research on new therapeutic targets. Recently established animal models of pancreatic cancer, including patient-derived tumor models and organoid models, are used for pre-clinical drug testing and biomarker discovery. These models have useful characteristics over conventional xenograft mouse models based on cell lines in preclinical studies, but still cannot accurately predict the clinical outcomes of new treatments and have not yet been broadly implemented in research. We employed pancreatic cancer organoid culture methods using the pancreatic cancer cell line S2-013, and performed pathological and immunohistochemical analyses to characterize tumor xenografts obtained from a mouse model implanted with S2-013 cell line-derived organoids. Serum levels of the pancreatic cancer tumor marker CA19-9 were measured by ELISA. We generated human pancreatic cancer organoids using a co-culture of S2-013 cells, human endothelial cells derived from human umbilical vein endothelial cells, and human mesenchymal stem cells, and established a mouse model with subcutaneously transplanted human pancreatic cancer organoids (S2-013-organoid model). Although blood clotting crater-like formation developed in the middle of subcutaneous xenografts in the S2-013-conventional model, created by subcutaneously injecting S2-013 cells into the right flank of nude mice, the size of xenografts in the S2-013-organoid model gradually increased without crater-like formation. Importantly, tumor xenografts obtained from the S2-013-organoid model exhibited a clinical human pancreatic cancer tissue-like cellular morphology, tissue architecture, and polarity, and actively formed cancer stroma containing mature blood vessels with the high expression of the vascular tight junction marker CD31. In subcutaneous xenografts of S2-013-conventional mice, no blood vessel density or widely expanding areas of necrotic regions were present. Consequently, serum levels of CA19-9 in the S2-013-organoid model correlated with tumor volumes. In addition, epithelial–mesenchymal transition, the conversion of epithelial cells to the mesenchymal phenotype, was observed in tumor xenografts of the S2-013-organoid model. The S2-013-organoid model provides tumor xenografts consisting of clinical human pancreatic cancer-like tissue formation with the effective development of vascularized stroma, and may be valuable for facilitating studies on pre-clinical drug testing and biomarker discovery.

Early Predictor Tool of Disease Using Label-Free Liquid Biopsy-Based Platforms for Patient-Centric Healthcare.

Simple SummaryWe proposed a comprehensive early prediction tool based on liquid biopsy for the label-free phenotypic analysis of cell clusters from clinical samples (n = 31). Our custom algorithm analysis, combined with a microfluidic-based tumor model, was designed to assess and stratify cancer patients in a label-free, cost-effective, and user-friendly way. Multiple quantitative phenotypic parameters (cluster size, thickness, roughness, and thickness per area) were derived from the profiling of the patient-derived cell clusters. Our platform could distinguish healthy donors from pretreatment cancer patients with high sensitivity (91.16 ± 1.56%) and specificity (71.01 ± 9.95%). In addition, the ratio of normalized gray value to cluster size (RGVS) parameter was significantly correlated to treatment duration and cancer stage. In conclusion, our patient-centric, early prediction tool will allow the prognosis of patients in a relatively less invasive manner, which can help clinicians identify diseases or indicate the need for new treatment strategies.Cancer cells undergo phenotypic changes or mutations during treatment, making detecting protein-based or gene-based biomarkers challenging. Here, we used algorithmic analysis combined with patient-derived tumor models to derive an early prediction tool using patient-derived cell clusters from liquid biopsy (LIQBP) for cancer prognosis in a label-free manner. The LIQBP platform incorporated a customized microfluidic biochip that mimicked the tumor microenvironment to establish patient clusters, and extracted physical parameters from images of each sample, including size, thickness, roughness, and thickness per area (n = 31). Samples from healthy volunteers (n = 5) and cancer patients (pretreatment; n = 4) could be easily distinguished with high sensitivity (91.16 ± 1.56%) and specificity (71.01 ± 9.95%). Furthermore, we demonstrated that the multiple unique quantitative parameters reflected patient responses. Among these, the ratio of normalized gray value to cluster size (RGVS) was the most significant parameter correlated with cancer stage and treatment duration. Overall, our work presented a novel and less invasive approach for the label-free prediction of disease prognosis to identify patients who require adjustments to their treatment regime. We envisioned that such efforts would promote the management of personalized patient care conveniently and cost effectively.

Longitudinal Monitoring of Intra-Tumoural Heterogeneity Using Optical Barcoding of Patient-Derived Colorectal Tumour Models.

Simple SummaryColorectal cancer (CRC) is the second most common cancer worldwide. Despite improvements in the clinical management of CRC, outcomes for those with metastatic disease remain extremely poor. One reason for this is tumour heterogeneity, which refers to the observation that each cell within complex tumour cell populations displays different genetic features and biological behaviours. Such tumour heterogeneity is known to impact treatment efficacy and promote tumour recurrence. Here, we present a multi-colour barcoding methodology that allows for different lineages of colorectal cancer cells to be identified and monitored, thus allowing for tumour heterogeneity to be quantified in real-time. We show that discrete cell lineages can be quantified by both fluorescence microscopy and flow cytometry. Using this approach, we show that the cell culture models that are traditionally used in cancer research display limited heterogeneity, whereas patient-derived organoids—which are generated from fresh tumour resections—more faithfully represent the heterogeneity observed in cancer patients.Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed “optical barcoding” (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of individual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.

Establishment of Patient-Derived Succinate Dehydrogenase-Deficient Gastrointestinal Stromal Tumor Models for Predicting Therapeutic Response.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract, with mutant succinate dehydrogenase (SDH) subunits (A-D) comprising less than 7.5% (i.e., 150-200/year) of new cases annually in the United States. Contrary to GISTs harboring KIT or PDGFRA mutations, SDH-mutant GISTs affect adolescents/young adults, often metastasize, and are frequently resistant to tyrosine kinase inhibitors (TKI). Lack of human models for any SDH-mutant tumors, including GIST, has limited molecular characterization and drug discovery. We describe methods for establishing novel patient-derived SDH-mutant (mSDH) GIST models and interrogated the efficacy of temozolomide on these tumor models in vitro and in clinical trials of patients with mSDH GIST. Molecular and metabolic characterization of our patient-derived mSDH GIST models revealed that these models recapitulate the transcriptional and metabolic hallmarks of parent tumors and SDH deficiency. We further demonstrate that temozolomide elicits DNA damage and apoptosis in our mSDH GIST models. Translating our in vitro discovery to the clinic, a cohort of patients with SDH-mutant GIST treated with temozolomide (n = 5) demonstrated a 40% objective response rate and 100% disease control rate, suggesting that temozolomide represents a promising therapy for this subset of GIST. We report the first methods to establish patient-derived mSDH tumor models, which can be readily employed for understanding patient-specific tumor biology and treatment strategies. We also demonstrate that temozolomide is effective in patients with mSDH GIST who are refractory to existing chemotherapeutic drugs (namely, TKIs) in clinic for GISTs, bringing a promising treatment option for these patients to clinic.See related commentary by Blakely et al., p. 3.

M6A Modification-Mediated DUXAP8 Regulation of Malignant Phenotype and Chemotherapy Resistance of Hepatocellular Carcinoma Through miR-584-5p/MAPK1/ERK Pathway Axis.

Hepatocellular carcinoma (HCC) has a poor prognosis due to its high malignancy, rapid disease progression, and the presence of chemotherapy resistance. Long-stranded non-coding RNAs (lncRNAs) affect many malignant tumors, including HCC. However, their mechanism of action in HCC remains unclear. This study aimed to clarify the role of DUXAP8 in regulating the malignant phenotype and chemotherapy resistance in HCC. Using an in vivo xenograft tumor model, the regulatory functions and mechanisms of lncRNA DUXAP8 in the progression and response of HCC to chemotherapy were explored. It was found that DUXAP8 was significantly upregulated in a patient-derived xenograft tumor model based on sorafenib treatment, which is usually associated with a relatively poor prognosis in patients. In HCC, DUXAP8 maintained its upregulation in the expression by increasing the stability of m6A methylation-mediated RNA. DUXAP8 levels were positively correlated with the proliferation, migration, invasion, and chemotherapy resistance of HCC in vivo and in vitro. In the mechanistic study, it was found that DUXAP8 competitively binds to miR-584-5p through a competing endogenous RNA (ceRNA) mechanism, thus acting as a molecular sponge for miR-584-5p to regulate MAPK1 expression, which in turn activates the MAPK/ERK pathway. These findings can provide ideas for finding new prognostic indicators and therapeutic targets for patients with HCC.