Patient-derived Organoid Models Research Articles

Combining TIGIT blockade with IL-15 stimulation is a promising immunotherapy strategy for lung adenocarcinoma.

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immune checkpoint molecule that suppresses CD8+ T-cell function in cancer. However, the expression profile and functional significance of TIGIT in the immune microenvironment of lung adenocarcinoma (LUAD) remain elusive. Interleukin (IL)-15 has emerged as a promising candidate for enhancing CD8+ T-cell mediated tumour eradication. Exploring therapeutic strategies that combine IL-15 with TIGIT blockade in LUAD is warranted. We investigated the regulatory network involving coinhibitory TIGIT and CD96, as well as costimulatory CD226 in LUAD using clinical samples. The potential role of TIGIT in regulating the pathogenesis of LUAD was addressed through a murine model with transplanted tumours constructed in Tigit-/- mice. The therapeutic strategy that combines TIGIT blockade with IL-15 stimulation was verified using a transplanted tumour murine model and a patient-derived organoid (PDO) model. The frequency of TIGIT+ CD8+ T cells was significantly increased in LUAD. Increased TIGIT expression indicated poorer prognosis in LUAD patients. Furthermore, the effector function of TIGIT+ CD8+ tumour-infiltrating lymphocytes (TILs) was impaired in LUAD patients and TIGIT inhibited antitumour immune response of CD8+ TILs in tumour-bearing mice. Mechanistically, IL-15 enhanced the effector function of CD8+ TILs but stimulated the expression of TIGIT on CD8+ TILs concomitantly. The application of IL-15 combined with TIGIT blockade showed additive effects in enhancing the cytotoxicity of CD8+ TILs and thus further increased the antitumour immune response in LUAD. Our findings identified TIGIT as a promising therapeutic target for LUAD. LUAD could benefit more from the combined therapy of IL-15 stimulation and TIGIT blockade.

Establishment of Patient-Derived Organoid Model from Hydrothorax and Ascites

Organoids are miniaturized organs cultured in vitro, exhibiting self-renewal and self-organization capabilities. In comparison to traditional 2D cell cultures, organoids can reproduce the spatial structure and functionality resembling corresponding tissues or organs, providing highly physiologically relevant in vitro systems. Consequently, they can be utilized for simulating disease occurrence, testing the tumor drug sensitivity and co-culturing with other cells. This study aims to establish a Patient-Derived Organoid (PDO) model using hydrothorax and ascites samples. To ensure cell viability, it is essential to store the collected malignant fluid at 4°C. Upon arrival at the laboratory, the fluid should be divided into centrifuge tubes for subsequent centrifugation, allowing the collection of cell precipitates. These cell precipitates need to be washed multiple times with DPBS until the supernatant appears clear. If the pellet contains red blood cells, they should be lysed, followed by cell counting. Depending on the desired seeding density, cells should be resuspended in Matrigel and plated as domes in a culture plate. The plate should then be inverted and placed in a 37°C incubator for 30 minutes to allow the Matrigel to solidify. Once solidified, complete culture medium should be added, and the culture plate should be placed in a 37°C incubator for further incubation. Organoid growth can be monitored using microscopy to assess their development and progression. Once the organoids have reached a stable growth state, immunohistochemistry can be performed to identify specific cell types or markers within the organoids. Furthermore, the drug responsiveness of the organoids can be evaluated through cell viability assays, enabling researchers to determine the effects of different drugs or organoid viability and function.

Genetic Alterations of NF-κB and Its Regulators: A Rich Platform to Advance Colorectal Cancer Diagnosis and Treatment.

Colorectal cancer (CRC) is the third leading cause of cancer mortality in the United States, with an estimated 52,000 deaths in 2023. Though significant progress has been made in both diagnosis and treatment of CRC in recent years, genetic heterogeneity of CRC-the culprit for possible CRC relapse and drug resistance, is still an insurmountable challenge. Thus, developing more effective therapeutics to overcome this challenge in new CRC treatment strategies is imperative. Genetic and epigenetic changes are well recognized to be responsible for the stepwise development of CRC malignancy. In this review, we focus on detailed genetic alteration information about the nuclear factor (NF)-κB signaling, including both NF-κB family members, and their regulators, such as protein arginine methyltransferase 5 (PRMT5), and outer dynein arm docking complex subunit 2 (ODAD2, also named armadillo repeat-containing 4, ARMC4), etc., in CRC patients. Moreover, we provide deep insight into different CRC research models, with a particular focus on patient-derived xenografts (PDX) and organoid models, and their potential applications in CRC research. Genetic alterations on NF-κB signaling components are estimated to be more than 50% of the overall genetic changes identified in CRC patients collected by cBioportal for Cancer Genomics; thus, emphasizing its paramount importance in CRC progression. Consequently, various genetic alterations on NF-κB signaling may hold great promise for novel therapeutic development in CRC. Future endeavors may focus on utilizing CRC models (e.g., PDX or organoids, or isogenic human embryonic stem cell (hESC)-derived colonic cells, or human pluripotent stem cells (hPSC)-derived colonic organoids, etc.) to further uncover the underpinning mechanism of these genetic alterations in NF-κB signaling in CRC progression. Moreover, establishing platforms for drug discovery in dishes, and developing Biobanks, etc., may further pave the way for the development of innovative personalized medicine to treat CRC in the future.

Autophagy activation is required for N6-methyladenosine modification to regulate ferroptosis in hepatocellular carcinoma

Background & aimsAlthough ferroptosis holds promise as a new strategy for treating hepatocellular carcinoma (HCC), there are several obstacles that need to be overcome. One major challenge is the lack of understanding about the mechanisms underlying ferroptosis. Additionally, while the m6A modification has been shown to regulate various forms of cell death, its role in regulating ferroptosis in HCC has been largely overlooked. Bridging this knowledge gap, our study aimed to elucidate the regulatory influence of m6A modification on HCC ferroptosis. MaterialsDot blot and EpiQuik m6A RNA Methylation Quantitative kit detected changes in overall m6A modification level during ferroptosis in HCC. MeRIP-qPCR and RIP-qPCR identified that the m6A modification of ATG5 mRNA was significant changed. BALB/c nude mice were used to construct xenograft tumor models to verify the phenotypes upon YTHDC2 silencing. In addition, patient-derived organoid models were used to demonstrate that induction of ferroptosis was an effective strategy against HCC. ResultsOur study has shown that inducing ferroptosis is a promising strategy for combatting HCC. Specifically, we have found a significant correlation between ferroptosis and high levels of m6A modification in HCC. Notably, we discovered that the elevation of ATG5 mRNA m6A modification mediated by WTAP is dependent on the reading protein YTHDC2. Importantly, inhibition of either WTAP or YTHDC2 effectively prevented ferroptosis and suppressed HCC development in both in vitro and in vivo models. ConclusionOur study revealed that WTAP upregulates ATG5 expression post-transcriptionally in an m6A-YTHDC2-dependent manner, thereby promoting the translation of ATG5 mRNA during ferroptosis in HCC. These findings have significant implications for the development of innovative and effective therapeutic approaches for HCC treatment.

Abstract C056: Characterizing the DNA methylome of gastric cancer among Hispanic/Latinos to advance cancer precision health equity

Abstract Gastric cancer (GC) is the second leading cause of cancer-related deaths worldwide. While overall incidence and mortality rates have dropped in recent decades, GC remains a significant cause of health disparities for many racial and ethnic minority groups in the US, including Hispanic/Latino Americans (HLAs). Despite a high minority cancer burden, few FDA-approved targeted therapies are available for GC. This can be partially explained by limited availability of cancer genome data and patient-derived models from racial/ethnic minority populations, hampering therapeutic target identification and drug efficacy studies. Our group has spearheaded the development of the University of California Minority Patient-Derived Xenograft Development and Trial Center (UCaMP) with the goal of addressing these critical issues for GC patient care. Thus far, we have characterized over 30 GC samples from racial/ethnic minorities with more than 60% of samples coming from HLAs. Genomic analyses have revealed a significantly higher prevalence of genomically stable GC (GS-GC) tumors among HLAs compared to TCGA, which is comprised of mostly NLWs. GS-GC is the most aggressive and treatment resistant of the four TCGA-defined molecular subtypes. While we have discovered genomic alterations in several therapeutically targetable pathways among our minority-patient GC tumors, GS-GC tumors remain largely devoid of targetable mutations. These observations have led us to assess the epigenome to inform therapeutic development. We performed reduced representation bisulfite sequencing on 15 GC tumor-adjacent normal pairs from our UCaMP cohort. We identified significantly hyper- and hypomethylated genes and performed overlaps with RNA expression data from TCGA stomach adenocarcinoma cases to identify epigenetically silenced and activated genes unique to each molecular subtype. For GS-GC samples, pathway enrichment analysis revealed significant enrichment of key cancer pathways among silenced genes for GS samples, including WNT/NOTCH, PI3K/Akt, focal adhesions and cell-extracellular matrix interactions, and pro-growth/anti-apoptotic signaling. Importantly, GS samples showed silencing of two PI3K/Akt pathway regulatory subunits, PIK3R2 and PIK3R5, suggesting aberrant signaling of this highly therapeutically targetable pathway despite lack of mutational alterations. This data has provided a basis for further testing of PI3K/Akt targeted inhibitors using our minority patient-derived organoid and xenograft models to treat GS-GC, a GC subtype for which there are currently few effective treatment options and is associated with significant health disparities for Hispanic/Latinos. Citation Format: Nicole B. Halmai, Hongyong Zhang, Paul C. Lott, Ana Estrada-Florez, Ted W. Toal, Javier Torres, UCaMP Consortium, Luis G. Carvajal-Carmona. Characterizing the DNA methylome of gastric cancer among Hispanic/Latinos to advance cancer precision health equity [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C056.

Abstract A071: Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma

Abstract Background Metastatic pancreas adenocarcinoma (PDAC) is highly lethal and minimally responsive to immunotherapy. Gamma delta T cells (gDTs) are a unique T cell subset found within the PDAC microenvironment. Animal models have shown that gDTs promote oncogenic progression and ablating gDT delays cancer progression and improves survival. Understanding the activity of immune effector cells against cancer organoids provides a scalable platform to understand the impact of activation and small molecule modulation to redirect gDT killing in PDAC. Methods Patient-derived organoids (PDOs) were generated from fresh tissue sampling and exapanded using a defined, serum-free media. Upon passaging, media was collected from PDAC PDOs. Allogeneic gDTs isolated from healthy donors were cultured in PDAC PDO conditioned media from 3 separate PDOs, in 3 separate biological replicates, and then subjected to bulk RNAseq. Allogeneic gDTs were isolated and cultured with PDAC PDOs at ratio of 5:1 (gDTs:PDAC PDOs) for 48 hours. After 48 hours, staining was performed for induction of apoptosis (cleaved caspase-3/7FITC). Images were acquired by high content imaging (Cytation 5, BioTek) and quantified for response across organoid populations. Based on RNAseq nominated candidates, low density lipoprotein (LDL) was added (10 ug/mL) as well as a small molecule inverse agonist of the Liver X Receptor (LXR) beta transcription factor (SR9243) to assess the ability of these factors to modulate gDT mediated PDAC killing. Results Exposure of gDTs to PDAC PDO secreted factors results in differential regulation of cholesterol handling, including upregulation of cholesterol efflux, and downregulation of cholesterol uptake and biosynthesis related genes in conditioned T cell populations. gDTs cause marked apoptosis in PDAC PDOs, including 40% staining of % organoid area versus negligible staining in control populations of 1.9% organoid area (effect size = 9.5, p<0.001). This response was directly compared to standard chemotherapy at the time of clinical resistance to FOLFIRINOX which conferred only 2% staining of mean organoid area (effect size = 0.11, NS). Given RNAseq data demonstrating cholesterol handling changes after exposure to PDAC secreted factors, we investigated LDL supplementation in our model. Supplementation of LDL significantly improves gDT mediated killing, with induction of apoptosis seen in 52% of the PDAC PDO area (p=0.003). This effect is mimicked by the small molecule SR9243 (an LXR inverse agonist), with 56% of PDAC PDO demonstrating apoptotic induction at 48 hours. Conclusions A PDAC PDO model system can be used to investigate gDT mediated PDAC killing when applied in low-volume medium throughput screening applications. Modulation of LXR activity and microenvironment LDL results in substantially increased immune-mediated killing of PDAC. This screening platform identifies potentially clinically actionable modulators of immune response to PDAC, and suggests potential future immunologically relevant targets that may improve the efficacy of immunotherapy. Citation Format: Johnathan D Ebben, David Turicek, Md Shahadat Hossan, Austin Stram, Ethan Lin, Nicholas Hess, Zachary Mayhew, Melissa A Kinney, Devin Burpee, Anikait Patel, Christian M Capitini, Jeremy D Kratz. Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A071.

Abstract B093: Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent

Abstract KRAS is the most frequently mutated oncoprotein in human cancers. Although long considered “undruggable”, recent breakthroughs in medicinal chemistry have led to FDA-approval of the KRAS(G12C) mutation-specific inhibitors sotorasib and adagrasib for the treatment of KRAS(G12C)-positive non-small cell lung cancer. However, illustrating the need to develop additional novel agents targeting mutated KRAS, the 5-year relative survival rate for pancreatic cancer, which is commonly associated with a variety of different KRAS mutations, is only 12% (all SEER stages) due to poor early detection and a lack of effective treatments. In particular, KRAS(G12D) is the most common KRAS mutation, being found in 37% of pancreatic ductal adenocarcinomas (PDACs), as well as in 12.5% of colorectal cancer and 4.9% of lung adenocarcinoma (LUAD) patients. MRTX1133 is a highly-selective, non-covalent KRAS(G12D) inhibitor that has recently entered a phase 1 clinical trial. However, here we show that exposure of KRAS(G12D)-mutated PDAC (PANC-1 and AsPC-1) and LUAD (SK-LU-1) cell lines and a patient-derived organoid (PDO) PDAC model (RPAN001) to MRTX1133 resulted in varying degrees of in vitro efficacy. Decreased downstream KRAS signaling in the form of reduced phospho-ERK1/2 levels was observed to rapidly recover within 24 hours of treatment with 500 nM MRTX1133. Moreover, this rebound coincided with increased expression of KRAS, NRAS andHRAS mRNAs. Concurrently, activation of the receptor tyrosine kinases (RTKs) EGFR and MET was observed in PANC-1 and SK-LU-1 cells that displayed innate resistance to MRTX1133, while the sensitive AsPC-1 cells showed no such RTK activation. These results suggest that targeting of both KRAS(G12D) and RTKs may be needed to treat KRAS(G12D) inhibitor-resistant cancers. To address this, we employed a novel hetero-bispecific CHAMP molecule, RNK08179, that simultaneously targets both KRAS(G12D) and HSP90, an RTK-regulating chaperone protein. RNK08179 treatment demonstrated a striking reduction in phospho-ERK1/2 levels, RTK activation and cell viability in MRTX1133-resistant PANC-1 and SK-LU-1 cells. Furthermore, similar efficacy was observed in the MRTX1133-resistant RPAN001 PDO model. In summary, RNK08179 displayed promising efficacy in cancer models harboring KRAS(G12D) by suppressing both mutated KRAS and HSP90-supported RTK signaling. Citation Format: Ines Pulido, Qiyue Luan, Chenghao Yin, Zimo Yang, Jinhua Lin, Yaya Wang, Yuetong Sun, Chuche Liu, Haoxin Zhou, Marek Massad, Ian Papautsky, Thomas L. Prince, Guoqiang Wang, Kevin P. Foley, Weiwen Ying, Takeshi Shimamura. Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B093.

Selective inhibition of CDK9 in triple negative breast cancer

Targeted therapy for triple-negative breast cancers (TNBC) remains a clinical challenge due to tumour heterogeneity. Since TNBC have key features of transcriptionally addicted cancers, targeting transcription via regulators such as cyclin-dependent kinase 9 (CDK9) has potential as a therapeutic strategy. Herein, we preclinically tested a new selective CDK9 inhibitor (CDDD11-8) in TNBC using cell line, patient-derived organoid, and patient-derived explant models. In vitro, CDDD11-8 dose-dependently inhibited proliferation (IC50 range: 281–734 nM), induced cell cycle arrest, and increased apoptosis of cell lines, which encompassed the three major molecular subtypes of TNBC. On target inhibition of CDK9 activity was demonstrated by reduced RNAPII phosphorylation at a CDK9 target peptide and down-regulation of the MYC and MCL1 oncogenes at the mRNA and protein levels in all cell line models. Drug induced RNAPII pausing was evident at gene promoters, with strongest pausing at MYC target genes. Growth of five distinct patient-derived organoid models was dose-dependently inhibited by CDDD11-8 (IC50 range: 272–771 nM), including three derived from MYC amplified, chemo-resistant TNBC metastatic lesions. Orally administered CDDD11-8 also inhibited growth of mammary intraductal TNBC xenograft tumours with no overt toxicity in vivo (mice) or ex vivo (human breast tissues). In conclusion, our studies indicate that CDK9 is a viable therapeutic target in TNBC and that CDDD11-8, a novel selective CDK9 inhibitor, has efficacy in TNBC without apparent toxicity to normal tissues.

IMMU-08. EOSINOPHILS ARE IMPORTANT MODULATORS OF AN IMMUNE-RESPONSIVE TUMOR-MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Eosinophils, bone marrow-derived granulocytes involved in allergic asthma and autoimmunity, have attracted increasing attention due to their assumed role in effective responses to immune checkpoint blockade in some extracranial tumors. However, the role of this rare type of immune cells in immunologically cold tumors such as glioblastoma (GBM) has not been studied so far. When quantifying immune infiltrates and levels of 30 immune-relevant cytokines in 60 matched pairs of human primary and recurrent GBM, strongest correlations (r= 0.71-0.93) were observed for the eosinophil-associated cytokines Eotaxin and Il-5 as well as for TNFa, IFNg and Il2 which are important cytokines for cytotoxic T cell responses. A combined score of these cytokines (Eo score) turned out to be an independent prognostic marker for improved overall survival of GBM patients. Furthermore, multicolor immunofluorescent staining revealed that Eo score high GBM are significantly higher infiltrated by effector T cells and show a significantly lower infiltration with anti-inflammatory M2-like tumor-associated macrophages / microglia cells (M2-TAM) in recurrent GBM. Flowcytometry analysis of GBM-derived single cell suspensions confirmed a positive correlation of higher eosinophil numbers with higher numbers of tumor-infiltrating T cells, while an inverse relation between eosinophils and monocytes could be demonstrated in the TCGA data set. Spatial transcriptomic data of human GBM confirmed that areas with a higher eosinophil infiltration showed higher numbers of T cells and pro-inflammatory M1-TAM, lower numbers of M2-TAM and a remarkable vessel normalization. Finally, in a newly developed patient-derived GBM organoid model we could corroborate the functional impact of eosinophils on the tumor-microenvironment. An increase of eosinophil numbers in these patient avatars resulted in a significantly decreased number of M2-TAM. Altogether, tumor-infiltrating eosinophils seem to be a novel type of immune-modulator in GBM and may enhance immune responses by shaping the tumor niche from an immunosuppressive to an immunepermissive microenvironment.

MODL-15. ESTABLISHMENT OF ORGANOID MODELS OF PATIENT-DERIVED GH-PRODUCING PITUITARY ADENOMA

Abstract BACKGROUND Growth hormone-producing pituitary adenomas (GHomas) reveal unfavorable clinical course due to systemic complications such as excessive sweating, impaired glucose tolerance, and hypertension, in addition to its characteristic facial appearance. For GHoma, pharmacological treatments including somatostatin analogs and dopamine agonists are widely performed, however, a subset of case reveals dismal prognosis. We need to elucidate molecular mechanisms contributing to GHoma growth, however, experimental models of GHoma such as cell line and animal model are quietly limited. OBJECTIVE In recent years, organoid models using 3D-culture technologies have gained attention as useful research models for various types of cancers. This study aims to establish a novel organoid model derived from GHoma patients. Using established GHoma organoid model, we analyzed their histological characteristics. METHODS Surgical specimens from five cases of GHoma were finely dissected to approximately 1mm and embedded in Matrigel basement membrane matrix. The specimens were then cultured under 37°C conditions in a 5% CO2 incubator using a medium supplemented with various growth factors. After 28 days of culture, formalin-fixed paraffin-embedded sections were prepared, and the histological similarity with patient tumors was examined. RESULTS Hematoxylin and eosin staining showed that organoid models consisted of acidophilic, granular cytoplasm and small round nuclei, maintaining a cell morphology similar with the original tumor. Immunohistochemical staining revealed positive expression of growth hormone, Pit-1, and CAM5.2, which are specific markers for GHoma. CONCLUSIONS We successfully established a patient-derived organoid model of GHoma that retains histological and endocrinological characteristics. We believe that this model could be a valuable research model for elucidating the molecular mechanisms of GHoma and developing novel treatment approaches targeting important molecular abnormalities. Additionally, in clinical settings, this model might be useful for predicting the responsiveness to drug treatment and selecting appropriate therapies for individual patients.

Dual Targeting of ROR1 and BTK Augments the Anti-Lymphoma Activity in Mantle Cell Lymphoma

Background Mantle cell lymphoma (MCL) is a rare and aggressive subtype of non-Hodgkin lymphoma. Development of BTK inhibitors (BTKis) and CAR-T cells revolutionized the treatment paradigm for the patients with MCL. However, this aggressive B-cell lymphoma frequently relapses leading to poor survival. Therefore, novel therapies are in urgent need to overcome the therapeutic resistance. ROR1 is normally expressed during embryonic development but is usually absent or expressed at low levels in adult tissues. However, re-expression of ROR1 was found in various types of cancer, especially in cancer stem/tumor-initiating cells. ROR1 persistently activates oncogenic signaling pathways in the BCR/BTK-independent manner. Accordingly, we hypothesize that dual targeting of ROR1 and BTK may augment the therapeutic efficacy, and thus combination of monoclonal antibody zilovertamab (cirmtuzumab) targeting ROR1, and BTKis will overcome the therapeutic resistance and effectively kill the relapsed/refractory MCL cells. Methods A panel of MCL cell lines and primary patient samples were used in this study. ROR1 expression levels in MCL cell lines and primary MCL samples were determined by flow cytometry and western blotting. For establishment of patient-derived organoid (PDO), the patient samples or MCL cell from PDX tumors were resuspended in culture medium containing cytokine cocktails. The cell aggregates within 50% Matrigel were gently transferred into multiple-well plates as needed. The established PDOs were applied for drug sensitivity assays. In addition, a patient-derived xenograft (PDX) mouse model was also used for evaluating single agent and combination efficacy of BTKis and zilovertamab (Zilo). Cell viability assays were performed using CellTiter routine or 3D cell viability assay kit (Promega). Results We hypothesize dual targeting of both BTK and ROR1 signaling pathways will augment cytotoxic effect on the MCL cells including major proliferative and minor ROR1 +CD117 + population, whose survival is not dependent on BTK, in tumor mass. To test the hypothesis, we first analyzed the ROR1 expression in MCL cell lines, primary and PDX MCL cells using flow cytometry and immunoblotting analysis. Our results indicated that in comparison with normal PBMC, ROR1 expression were detected in all examined MCL cell lines including JeKo-1, Mino, Z-138, Granta519, JVM2, SP49, Maver-1 and Rec-1. To test whether p53 status relates to sensitivity of MCL cells to BTKi and Zilo, we examined cytotoxic effect of combination of BTKi and Zilo on MCL cell lines (1) harboring mutated TP53 (T P53 Mut): JeKo-1, JeKo-R, Mino, and Mino-R and (2) harboring wild-type TP53 ( TP53 WT): Z-138 and Maver-1, and PDO as well as PDX tumors derived from biopsies bearing either TP53 WT or TP53 Mut. Treatment with single BTKi at 2-10 µM (Ibrutinib, zanubrutinib, acalabrutinib or pirtobrutinib) significantly induced cytotoxicity in the TP53 MutMCL cells, and the cell death was remarkedly increased when BTKi was combined with Zilo at 25-50 µg/ml. In contrast, in MCL cell lines with TP53 WT, BTKi-Zilo combination did not significantly result in increase of cytotoxic effect compared to single agents. These results suggest that the genetic status of TP53 is important for the augmented cytotoxic effect of BTKi-Zilo combination on MCL cells. To further evaluate potentiated effect of BTKi-Zilo combination, we performed ex vivo viability assay using PDO platform. Typically, PDOs were treated with either single agent or combined regimens. Consistent with the data in MCL cell lines, BTKi-Zilo combination resulted in potentiated cytotoxic activity in the PDO models derived from naïve and relapsed patients harboring TP53 Mut. Furthermore, BTKi-Zilo combination displayed synergistic effect in a PDX model derived from a patient harboring TP53 Mut. Conclusion Dual targeting of BTK and ROR1 signaling pathways augmented efficacy selectively in preclinical MCL models with TP53 Mut. These data provide insights to develop tailored therapeutics to improve patient outcome for patients with TP53 mutation.

H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling.

Gastric cancer (GC) is one of the most common tumours in East Asia countries and is associated with Helicobacter pylori infection. H. pylori utilizes virulence factors, CagA and VacA, to up-regulate pro-inflammatory cytokines and activate NF-κB signaling. Meanwhile, the PIEZO1 upregulation and cancer-associated fibroblast (CAF) enrichment were found in GC progression. However, the mechanisms of PIEZO1 upregulation and its involvement in GC progression have not been fully elucidated. The CAF enrichment and clinical significance were investigated in animal models and primary samples. The expression of NF-κB and PIEZO1 in GC was confirmed by immunohistochemistry staining, and expression correlation was analysed in multiple GC datasets. GSEA and Western blot analysis revealed the YAP1-CTGF axis regulation by PIEZO1. The stimulatory effects of CTGF on CAFs were validated by the co-culture system and animal studies. Patient-derived organoid and peritoneal dissemination models were employed to confirm the role of the PIEZO1-YAP1-CTGF cascade in GC. Both CAF signature and PIEZO1 were positively correlated with H. pylori infection. PIEZO1, a mechanosensor, was confirmed as a direct downstream of NF-κB to promote the transformation from intestinal metaplasia to GC. Mechanistic studies revealed that PIEZO1 transduced the oncogenic signal from NF-κB into YAP1 signaling, a well-documented oncogenic pathway in GC progression. PIEZO1 expression was positively correlated with the YAP1 signature (CTGF, CYR61, and c-Myc, etc.) in primary samples. The secreted CTGF by cancer cells stimulated the CAF infiltration to form a stiffened collagen-enrichment microenvironment, thus activating PIEZO1 to form a positive feedback loop. Both PIEZO1 depletion by shRNA and CTGF inhibition by Procyanidin C1 enhanced the efficacy of 5-FU in suppressing the GC cell peritoneal metastasis. This study elucidates a novel driving PIEZO1-YAP1-CTGF force, which opens a novel therapeutic avenue to block the transformation from precancerous lesions to GC. H. pylori-NF-κB activates the PIEZO1-YAP1-CTGF axis to remodel the GC microenvironment by promoting CAF infiltration. Targeting PIEZO1-YAP1-CTGF plus chemotherapy might serve as a potential therapeutic option to block GC progression and peritoneal metastasis.

The potential of patient-derived organoids in precision medicine of biliary tract cancer

Chemotherapy resistance in biliary tract cancer (BTC) presents a major clinical hurdle. Ren etal.1 developed and characterized an extensive collection of BTC patient-derived organoid (PDO) models, enabling advanced investigation of chemotherapy response prediction.

KLHL29-mediated DDX3X degradation promotes chemosensitivity by abrogating cell cycle checkpoint in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype and accounts for approximately 15–20% of breast cancer cases. In this study, we identified KLHL29, which is an understudied member of the Kelch-like gene family, as a crucial tumor suppressor that regulates chemosensitivity in TNBC. KLHL29 expression was significantly downregulated in breast cancer tissues compared with adjacent normal tissues, and low levels of KLHL29 were associated with unfavorable prognoses. Ectopic KLHL29 suppressed, while depleting KLHL29 promoted, the growth, proliferation, migration, and invasion of TNBC. Mechanistically, KLHL29 recruited the CUL3 E3-ligase to the RNA-binding protein DDX3X, leading to the proteasomal degradation of the latter. This downregulation of DDX3X resulted in the destabilization of CCND1 mRNA and the consequent cell cycle arrest at G0/G1 phase. Remarkably, the DDX3X inhibitor RK33 combined with platinum-based chemotherapy can synergistically suppress TNBC that usually expresses low levels of KLHL29 and high levels of DDX3X using cancer cell-derived xenograft and patient-derived organoids models. Altogether, we uncovered the potential role for the KLHL29-DDX3X signaling cascade in the regulation of TNBC progression, thus providing a promising combination strategy for overcoming TNBC chemoresistance.

Unlocking the potential of organoids in cancer treatment and translational research: An application of cytologic techniques.

Patient-derived organoid models hold promise for advancing clinical cancer research, including diagnosis and personalized and precision medicine approaches, and cytology, in particular, plays a pivotal role in this process. These three-dimensional multicellular structures are heterogeneous, potentially maintain the cancer phenotype, and conserve the genomic, transcriptomic, and epigenomic patterns of the parental tumors. To ensure that only tumor tissue is used for organoid development, cytologic validation is necessary before initiating the process of organoid generation. Here, we explore the technology of tumor organoids and discuss the fundamental application of cytology as a simple and cost-effective approach toward organoid development. We also underscore the potential application of organoid development in drug efficacy studies for lung cancer and head and neck tumors. Additionally, we stress the importance of using fine-needle aspiration to generate tumoroids.

Cancer-associated fibroblasts impair the cytotoxic function of NK cells in gastric cancer by inducing ferroptosis via iron regulation

As the predominant immunosuppressive component within the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) inhibit Natural Killer cell (NK cell) activity to promote tumor progression and immune escape; however, the mechanisms of cross-talk between CAFs and NK cells in gastric cancer (GC) remain poorly understood. In this study, we demonstrate that NK cell levels are inversely correlated with CAFs abundance in human GC. CAFs impair the anti-tumor capacity of NK cells by inducing ferroptosis, a cell death process characterized by the accumulation of iron-dependent lipid peroxides. CAFs induce ferroptosis in NK cells by promoting iron overload; conversely, decreased intracellular iron levels protect NK cells against CAF-induced ferroptosis. Mechanistically, CAFs increase the labile iron pool within NK cells via iron export into the TME, which is mediated by the upregulated expression of iron regulatory genes ferroportin1 and hephaestin in CAFs. Moreover, CAF-derived follistatin like protein 1(FSTL1) upregulates NCOA4 expression in NK cells via the DIP2A-P38 pathway, and NCOA4-mediated ferritinophagy is required for CAF-induced NK cell ferroptosis. In a human patient-derived organoid model, functional targeting of CAFs using a combination of deferoxamine and FSTL1-neutralizing antibody significantly alleviate CAF-induced NK cell ferroptosis and boost the cytotoxicity of NK cells against GC. This study demonstrates a novel mechanism of suppression of NK cell activity by CAFs in the TME and presents a potential therapeutic approach to augment the immune response against GC mediated by NK cells.

Fisetin induces the upregulation of AKAP12 mRNA and anti-angiogenesis in a patient-derived organoid xenograft model

Colorectal cancer (CRC) is associated with high incidence and mortality rates. Targeted therapies for CRC cause various adverse effects, necessitating the development of novel approaches to control CRC progression. In this milieu, we investigated the anti-CRC effects of fisetin, a natural plant flavonoid. Cytotoxicity was performed in CRC patient-derived organoids (30 T and 33 T). Fisetin-induced tumor growth was evaluated in a CRC patient-derived organoid xenograft (PDOX) model. RNA sequencing, immunohistochemistry, and western blotting were performed subsequently. Fisetin significantly decreased organoid viability in a dose-dependent manner. In the PDOX model, fisetin significantly delayed tumor growth, showing a decrease in Ki-67 expression and the induction of apoptosis. In tumor tissues, four genes were identified as differentially expressed between the control and fisetin-treated groups. Among these, A-kinase anchoring protein 12 (AKAP12) level was significantly increased by fisetin treatment (fold change > 2, p < 0.05). Notably, fisetin significantly inhibited vascular endothelial growth factor (VEGF) and epithelial cell adhesion molecule (EpCAM) via upregulation of AKAP12. Our results demonstrate the upregulation of AKAP12 mRNA and inhibition of angiogenesis by fisetin as a therapeutic strategy against CRC.

Targeting LINC01607 sensitizes hepatocellular carcinoma to Lenvatinib via suppressing mitophagy

Lenvatinib is a standard therapy option for advanced hepatocellular carcinoma (HCC), but resistance limits clinical benefits. In this study, we identified inhibition of ROS levels and reduced redox status in Lenvatinib-resistant HCC. Integrating RNA-seq with unbiased whole-genome CRISPR-Cas9 screen analysis indicated LINC01607 regulated the P62 to enhance drug resistance by affecting mitophagy and antioxidant pathways. Underlying mechanisms were investigated both in vitro and in vivo. We initially confirmed that LINC01607, as a competing endogenous RNA (ceRNA) competing with mirRNA-892b, triggered protective mitophagy by upregulating P62, which reduced ROS levels and promoted drug resistance. Furthermore, LINC01607 was proved to resist oxidative stress by regulating the P62-Nrf2 axis, which transcriptionally regulated the expression of LINC01607 to form a positive feedback loop. Finally, silencing LINC01607 combined with Lenvatinib reversed resistance in animal and patient-derived organoid models. In conclusion, we proposed a novel mechanism of Lenvatinib resistance involving ROS homeostasis. This work contributed to understanding redox homeostasis-related drug resistance and provided new therapeutic targets and strategies for HCC patients.

Multifunctional CaCO3@Cur@QTX125@HA nanoparticles for effectively inhibiting growth of colorectal cancer cells

Colorectal cancer (CRC) is a major cause of cancer-related deaths in humans, and effective treatments are still needed in clinical practice. Despite significant developments in anticancer drugs and inhibitors, their poor stability, water solubility, and cellular membrane permeability limit their therapeutic efficacy. To address these issues, multifunctional CaCO3 nanoparticles loaded with Curcumin (Cur) and protein deacetylase (HDAC) inhibitor QTX125, and coated with hyaluronic acid (HA) (CaCO3@Cur@QTX125@HA), were prepared through a one-step gas diffusion strategy. Dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) showed that CaCO3@Cur@QTX125@HA nanoparticles have uniform spherical morphology and elemental distribution, with diameters around 450 nm and a Zeta potential of − 8.11 mV. The controlled release of Cur from the nanoparticles was observed over time periods of 48 h. Cellular uptake showed that CaCO3@Cur@QTX125@HA nanoparticles were efficiently taken up by cancer cells and significantly inhibited their growth. Importantly, CaCO3@Cur@QTX125@HA nanoparticles showed specific inhibitory effects on CRC cell growth. Encouragingly, CaCO3@Cur@QTX125@HA nanoparticles successfully internalized into CRC patient-derived organoid (PDO) models and induced apoptosis of tumor cells. The multifunctional CaCO3@Cur@QTX125@HA nanoparticles hold promise for the treatment of CRC.

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing.

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.