Tumor Organoids Research Articles

DDDR-22. USE OF BIOPSY-DERIVED TUMOR ORGANOIDS FOR PERSONALIZED DRUG TESTING IN RECURRENT GLIOBLASTOMA

Abstract To date, there are no effective systemic treatment options for recurrent glioblastoma (GBM). Furthermore, often surgical resection is not possible and tumors have acquired an increased chemo- or radioresistance. Patient-derived tumor organoids (PDTOs) provide a new exciting tool to test individual treatment responses. Aim of this study was to assess the feasibility of a personalized PDTO-based drug testing even from rare material such as stereotactic biopsies. PDTOs were prepared based on single cell suspensions from tumor material obtained either from open GBM resections (reference data set, n = 37) or from stereotactic biopsies (n = 8). Drug response curves (6-9 doses) were performed for 9 drugs including commonly used drugs such as temozolomide, lomustine, etoposide and temsirolimus. Viability was measured using CellTiterGlo (Promega) to assess half-maximal inhibitory concentrations (IC50). Medical records were reviewed for survival and molecular information such as the MGMT methylation status. Drug testing on PDTOs from open resections allowed to adjust the test range for each drug. Additionally, a significant association between increased temozolomide sensitivity of PDTOs and both, MGMT methylation and increased survival was observed. However, in 59% of the cases, no sensitivity to any of the drugs was seen, and only 17% were highly sensitive to more than one drug. Subsequent drug testing on PDTOs from biopsies of recurrent GBM revealed sensitivity in 3/8 cases against one (n=2) or two (n=1) of the drugs. However, sensitivity against drugs varied substantially in a patient-individual manner. We successfully developed a workflow to test drug sensitivity on PDTO even from low starting material obtained from stereotactic biopsies. Association of clinical outcome and MGMT methylation with temozolomide sensitivity corroborated the strength of this approach. Furthermore, patient-individual treatment responses strongly suggest for a future personalized drug testing.

IMMU-46. CROSS-TALK OF INNATE IMMUNE CELLS IN GLIOBLASTOMA PATHOPHYSIOLOGY: UNDERSTANDING NLRP3 AND NLRP12

Abstract Glioblastoma (GBM) are malignant gliomas with an average survival of less than 14 months after diagnosis, despite surgery, chemotherapy, and radiotherapy. Tumor survival is aided by the heterogeneous cell populations in the tumor microenvironment. Two crucial innate immune cells of the brain, namely microglia and macrophages, favor a tumor-promoting microenvironment IN GBM. The nucleotide-binding domain leucine-rich repeat-containing receptor (NLR) family in astrocytes and microglia cells recognizes pathogen- and damage-associated molecular patterns that induce inflammation. NLRP3, upon activation, leads to cleavage and activation of Caspase-1, causing the proteolytic cleavage-mediated activation and conversion of pro-IL-1ß and pro-IL-18 to IL-1ß and IL-18, leading to pyroptosis. NLRP12 downregulates inflammatory responses in microglia and macrophages by regulating the NF-kB pathway. NLRP3 and NLRP12 have tumor-promoting and anti-tumour functions in various cancers. NLRP12 is a prognostic marker for glioblastoma, and its increased expression correlates with poor survival in GBM patients. However, the cellular and molecular mechanisms of NLRP3 and NLRP12 in GBM pathophysiology are largely unknown. This study aims to understand the effect of NLRP3 and NLRP12 expression in GBM, to interpret the pathophysiology, and to help develop future targeted therapeutic interventions. To understand the baseline expression of NLRP3 and NLRP12 in cell lines and patient-derived GBM cells, PCR, western blotting, and Immunocytochemistry were performed comparing WT and nlrp3-/- and nlrp12-/- cells. Differential expression of NLRP3 and NLRP12 in patient-derived cells can be compared to GBM, astrocyte, and microglia cell lines. This may be the underlying cause for intra and inter-tumor heterogeneity and resultant differential therapy outcomes. To understand cell-to-cell communication in GBM-like tumor microenvironments, heterocellular 3D organoids, and patient-derived tumor organoids were developed. Cell viability in 3D patient-derived tumor organoids decreased by up to 10% with Doxorubicin and 40% with Carboplatin treatment decreases compared to untreated controls. This suggests the scope of developing drugs targeting inflammation regulatory genes.

DDDR-23. DECODING GLIOBLASTOMA DRUG RESISTANCE DYNAMICS USING NOVEL MICROFLUIDIC EX VIVO MODELS

Abstract Glioblastoma (GBM) is clinically challenging due to its aggressive nature and limited treatment options. Current standard of care involves surgical resection and chemoradiation with temozolomide (TMZ). GBM, however, often returns post-treatment fueled by its complex biology and the presence of resilient GBM stem cells. Compounding the issue is the blood-brain barrier, which poses a formidable obstacle to drug delivery into the brain. Understanding the molecular dynamics post-therapy within the brain, influenced by the tissue microenvironment, is key for developing more effective therapeutic strategies. Unfortunately, existing research models have fallen short in capturing the complexity of GBM and its response to treatment. While animal models have been a mainstay in GBM research, their ability to predict human responses has been called into question, highlighted by the disappointing results seen in clinical trials. We are therefore advancing bioprinting-based approaches for modeling various components of the tumor microenvironment to gain insight into mechanisms of drug resistance. We report on the development of three novel microfluidic platforms to investigate and interrogate GBM biology. First, we have developed a novel microfluidic model of GBM, termed GlioFlow-3D, produced through a combination of 3D printing methods. GlioFlow-3D mimics specific aspects of the perivascular niche, including protection from circulating TMZ. Second, we have adapted GlioFlow-3D as part of a platform to investigate advanced ultrasound techniques for delivering novel therapeutics into the human brain. Third, we are developing a novel approach aimed at exploring the relationship between the brain extracellular matrix and tumoral cells using bioprinting of living tumor organoids obtained from patients with a history of recurrence. By integrating bioprinting technologies with patient-derived models to create novel ex vivo platforms, we aim to obtain new insights into the intricate dynamics of drug resistance in GBM, ultimately paving the way for more effective therapeutic strategies. Funding: The Brain Tumor Center Research Award (UCGNI) The ARC Project - CCHMC and UC joint Research Award

TMIC-77. ELUCIDATING THE ROLE OF PTPRZ1 SIGNALING IN DRIVING GLIOBLASTOMA PROGRESSION USING THE NOVEL HUMAN ORGANOID TUMOR TRANSPLANTATION SYSTEM

Abstract Glioblastoma (GBM), the most malignant and aggressive primary brain tumor, is driven by both intrinsic genetic mutations and extrinsic factors from the tumor microenvironment (TME). GBM manipulates the TME to enhance its survival and proliferation through mechanisms such as forming functional synapses with adjacent normal cells and secreting immunosuppressive factors. Our laboratory has developed a novel human organoid tumor transplantation (HOTT) model, which allows for the interrogation of tumor-normal cell crosstalk at a molecular level and facilitates the dissection of specific signaling pathways that drive tumor progression and maintenance. Using single-cell transcriptomics on human GBM HOTTs, we identified the PTN-PTPRZ1 ligand-receptor interaction as a critical mediator of tumor-microenvironment communication. PTPRZ1, a receptor tyrosine phosphatase, has been previously implicated in glioma cell migration, and our data further elucidate its role in modulating tumor behavior. Specifically, we demonstrated that knockdown of PTPRZ1 within the TME of human cortical organoids significantly enhances glioma cell migration and inhibits differentiation, underscoring the essential role of PTPRZ1 in maintaining the tumor’s invasive phenotype. Further pharmacological inhibition of PTPRZ1’s catalytic activity with a small molecule revealed that these effects on cell fate determination are independent of its phosphatase activity. This suggests that PTPRZ1 exerts critical non-catalytic functions that are pivotal for GBM progression. To further investigate these non-catalytic mechanisms, we propose utilizing single-cell transcriptomics coupled with proximity labeling and proteomics. This approach will allow us to comprehensively map PTPRZ1-mediated signaling networks and elucidate their impact on tumor biology, potentially unveiling novel therapeutic targets. Our findings revealed a novel axis of GBM biology mediated by PTPRZ1 and highlighted the importance of considering microenvironmental interactions in the development of effective therapeutics for GBM.

Culturally responsive strategies and practical considerations for live tissue studies in Māori participant cohorts

IntroductionIndigenous communities globally are inequitably affected by non-communicable diseases such as cancer and coronary artery disease. Increased focus on personalized medicine approaches for the treatment of these diseases offers opportunities to improve the health of Indigenous people. Conversely, poorly implemented approaches pose increased risk of further exacerbating current inequities in health outcomes for Indigenous peoples. The advancement of modern biology techniques, such as three-dimensional (3D) in vitro models and next generation sequencing (NGS) technologies, have enhanced our understanding of disease mechanisms and individualized treatment responses. However, current representation of Indigenous peoples in these datasets is lacking. It is crucial that there is appropriate and ethical representation of Indigenous peoples in generated datasets to ensure these technologies can be used to maximize the benefit of personalized medicine for Indigenous peoples.MethodsThis project discusses the use of 3D tumor organoids and single cell/nucleus RNA sequencing to study cancer treatment responses and explore immune cell roles in coronary artery disease. Using key pillars from currently available Indigenous bioethics frameworks, strategies were developed for the use of Māori participant samples for live tissue and sequencing studies. These were based on extensive collaborations with local Māori community, scientific leaders, clinical experts, and international collaborators from the Broad Institute of MIT and Harvard. Issues surrounding the use of live tissue, genomic data, sending samples overseas and Indigenous data sovereignty were discussed.ResultsThis paper illustrates a real-world example of how collaboration with community and the incorporation of Indigenous worldviews can be applied to molecular biology studies in a practical and culturally responsive manner, ensuring fair and equitable representation of Indigenous peoples in modern scientific data.

Cancer-associated fibroblasts shape early myeloid cell response to chemotherapy-induced immunogenic signals in next generation tumor organoid cultures

BackgroundPatient-derived colorectal cancer (CRC) organoids (PDOs) solely consisting of malignant cells led to major advances in the understanding of cancer treatments. Yet, a major limitation is the absence of cells...

Specific ECM degradation potentiates the antitumor activity of CAR-T cells in solid tumors.

Although major progress has been made in the use of chimeric antigen receptor (CAR)-T-cell therapy for hematological malignancies, this method is ineffective against solid tumors largely because of the limited infiltration, activation and proliferation of CAR-T cells. To overcome this issue, we engineered CAR-T cells with synthetic Notch (synNotch) receptors, which induce local tumor-specific secretion of extracellular matrix (ECM)-degrading enzymes at the tumor site. SynNotch CAR-T cells achieve precise ECM recognition and robustly kill targeted tumors, with synNotch-induced enzyme production enabling the degradation of components of the tumor ECM. In addition, this regulation strongly increased the infiltration of CAR-T cells and the clearance of solid tumors, resulting in tumor regression without toxicity in vivo. Notably, synNotch CAR-T cells also promoted the persistent activation of CAR-T cells in patient-derived tumor organoids. Thus, we constructed a synthetic T-cell system that increases the infiltration and antitumor function of CAR-T cells, providing a strategy for targeting ECM-rich solid tumors.

Feasibility analysis of using patient-derived tumour organoids for treatment decision guidance in locally advanced head and neck squamous cell carcinoma

BackgroundCurrent treatment for head and neck squamous cell carcinoma (HNSCC) involves surgery, radiotherapy, and chemotherapy. Despite aggressive multimodal approaches, tumour recurrence occurs in 40–60 % of cases, leading to poor survival outcomes. HNSCC lacks common genetic drivers for tailored therapies, and reliable biomarkers for treatment selection are scarce. We investigated the procedural requirements for incorporating drug- and radiosensitivity screens in patient-derived organoids (PDOs) within a clinical trial framework. Patients and methodsFresh tumour samples (N = 198) from 186 HNSCC patients were included. Success rates of organoid establishment were correlated with clinical and procedural parameters. Timelines for establishment of PDO cultures were determined, and their long-term growth potential assessed by serial passaging. Additionally, we conducted whole exome sequencing on matched tumour-organoid pairs. Three PDO models were employed to establish radiosensitivity assays. ResultsIn total, PDO models displaying histomorphological features and genomic alterations of parental tumours were successfully established for 35 % of patient tumours. Success rates rose to 77 % for samples with a tumour cell content of 30 % or higher. Advanced patient age, prior radiotherapy, and delays in tissue processing were identified as negative predictors for engraftment. The estimated time interval needed for screens was compatible with PDO-guided selection of curative-intent radiotherapy regimens. ConclusionsOur findings suggest that with high-quality samples and efficient tissue processing, PDO screens can be successfully performed in 77 % of HNSCC patients. Given the procedural challenges involved, future clinical trials aiming to the utility of PDOs for guiding treatment decisions should consider implementing centralised PDO screening.

High-throughput solutions in tumor organoids: From culture to drug screening.

Tumor organoids have emerged as an ideal in vitro model for patient-derived tissues, as they recapitulate the characteristics of the source tumor tissue to a certain extent, offering the potential for personalized tumor therapy and demonstrating significant promise in pharmaceutical research and development. However, establishing and applying this model involves multiple labor-intensive and time-consuming experimental steps and lacks standardized protocols and uniform identification criteria. Thus, high-throughput solutions are essential for the widespread adoption of tumor organoid models. This review provides a comprehensive overview of current high-throughput solutions across the entire workflow of tumor organoids, from sampling and culture to drug screening. Furthermore, we explore various technologies that can control and optimize single-cell preparation, organoid culture, and drug screening with the ultimate goal of ensuring the automation and high efficiency of the culture system and identifying more effective tumor therapeutics.

Tumor organoids improve mutation detection of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) presents challenges in detecting somatic mutations due to its complex cellular composition. This study investigated the utility of patient-derived organoids (PDOs) to overcome these obstacles and enhance somatic mutation identification. Surgically resected PDAC tumors and their paired PDOs from 21 patients were examined. Whole-exome sequencing (WES) of tumor tissue, organoids, and peripheral blood mononuclear cells was performed to identify somatic mutations. Our findings demonstrate that PDOs retained about 80% of the somatic mutations from the original tumors, showing high concordance in mutation types. PDOs exhibited increased tumor purity and uncovered key driver mutations, aiding in identifying clinically relevant genomic alterations. Moreover, eight cycles of FOLFIRINOX treatment did not significantly alter the mutational landscape at the DNA level, indicating the stability of the mutational profile after therapeutic pressure in patients. In conclusion, PDOs are potentially important tools for exploring the somatic mutational landscape of PDAC. While they can reveal mutations that may be challenging to detect through traditional biopsy sequencing due to the inherently low tumor purity of PDAC, it is important to note that PDOs may not always fully recapitulate all mutations found in primary tumors. Despite this limitation, PDOs can still offer critical insights into the genomic complexities of PDAC, which is crucial for the development of personalized vaccines and therapies for this disease.

Single-cell chemoproteomics identifies metastatic activity signatures in breast cancer.

Protein activity state, rather than protein or mRNA abundance, is a biologically regulated and relevant input to many processes in signaling, differentiation, development, and diseases such as cancer. While there are numerous methods to detect and quantify mRNA and protein abundance in biological samples, there are no general approaches to detect and quantify endogenous protein activity with single-cell resolution. Here, we report the development of a chemoproteomic platform, single-cell activity-dependent proximity ligation, which uses automated, microfluidics-based single-cell capture and nanoliter volume manipulations to convert the interactions of family-wide chemical activity probes with native protein targets into multiplexed, amplifiable oligonucleotide barcodes. We demonstrate accurate, reproducible, and multiplexed quantitation of a six-enzyme (Ag-6) panel with known ties to cancer cell aggressiveness directly in single cells. We further identified increased Ag-6 enzyme activity across breast cancer cell lines of increasing metastatic potential, as well as in primary patient-derived tumor cells and organoids from patients with breast cancer.

Transport in cellular aggregates described by fluctuating hydrodynamics

Biological functionality of cellular aggregates is largely influenced by the activity and displacements of individual constituent cells. From a theoretical perspective this activity can be characterized by hydrodynamic transport coefficients of diffusivity and conductivity. Motivated by the clustering dynamics of bacterial microcolonies we propose a model of active multicellular aggregates and use recently developed macroscopic fluctuation theory to derive a fluctuating hydrodynamics for this model system. Both semianalytic theory and microscopic simulations show that the hydrodynamic transport coefficients are affected by nonequilibrium microscopic parameters and significantly decrease inside of the clusters. We further find that the Einstein relation connecting the transport coefficients and fluctuations breaks down in the parameter regime where the detailed balance is not satisfied. This study offers valuable tools for experimental investigation of hydrodynamic transport in other systems of cellular aggregates such as tumor spheroids and organoids. Published by the American Physical Society 2024

Identification of HSPG2 as a bladder pro-tumor protein through NID1/AKT signaling

PurposeHeparan sulfate proteoglycans (HSPGs) are complex molecules found on the cell membrane and within the extracellular matrix, increasingly recognized for their role in tumor progression. This study aimed to investigate the involvement of Heparan sulfate proteoglycan 2 (HSPG2) in the progression of bladder cancer.MethodsWe identified HSPG2 as a promoter of bladder tumor progression using single-cell RNA sequencing and transcriptome analysis of sequencing data from seven patient samples obtained from the Gene Expression Omnibus (GEO) database (GSE135337). Transcript profiles of 28 normal tissues and 407 bladder tumor tissues were analyzed for HSPG2 expression and survival outcomes using the Sanger tools and cBioPortal databases. HSPG2-overexpressing T24 and Biu-87 cell lines were generated, and cell proliferation and migration were assessed using CCK-8 and Transwell assays. Western blotting and immunostaining were performed to evaluate the activation of Nidogen-1 (NID1)/protein kinase B (AKT) signaling. Mouse models with patient-derived tumor organoids (HSPG2high and HSPG2low) were established to assess anticancer effects.ResultsOur results demonstrated a marked upregulation of HSPG2 in malignant bladder tumors, which correlated significantly with poor patient prognosis. HSPG2 overexpression consistently enhanced bladder tumor cell proliferation and conferred chemotherapy resistance, as shown in both in vitro and in vivo experiments. Mechanistically, HSPG2 upregulated NID1 expression, leading to the activation of the AKT pro-survival signaling pathway and promoting sustained tumor growth in bladder cancer.ConclusionThis study highlights the critical pro-tumor role of HSPG2/NID1/AKT signaling in bladder cancer and suggests its potential as a therapeutic target in clinical treatment.

Expert consensus on culture of patient derived tumor organoids and organoids based drug sensitivity testing

The precision treatment of cancer has consistently been a significant issue in clinical management as well as medical research. With the rapid development and advances towards clinical application of organoids technology, the use of organoids derived from both normal and cancer tissues for pathophysiology studies, treatment regimen development, and drug screening has become increasingly prevalent. This document compiles insights from dozens of nationwide experts and scholars in the field of tumor organoids drug sensitivity testing. It discusses the entire process of tumor organoids drug sensitivity testing, with the intent to provide expert guidance for laboratories interested in conducting clinical testing and application research related to cancer precision therapy.

Inhibition of KDM4A restricts SQLE transcription and induces oxidative stress imbalance to suppress bladder cancer

In clinical practice, the limited efficacy of standard comprehensive therapy for advanced bladder cancer and the lack of targeted treatment options are well recognized. Targeting abnormal epigenetic modifications in tumors has shown considerable potential in cancer therapy. Through drug screening in tumor organoids, we identified that ML324, a histone lysine demethylase 4A (KDM4A) inhibitor, exhibits potent antitumor effects in both in vitro and in vivo cancer models. Mechanistically, Kdm4a demethylates H3K9me3, leading to chromatin opening and increased accessibility of Gabpa to the squalene epoxidase (Sqle) gene promoter, resulting in transcriptional activation. Inhibition of Kdm4a downregulates Sqle transcription, blocking cholesterol synthesis and causing squalene (SQA) accumulation. This process induces reactive oxygen species (ROS) clearance and suppresses JNK/c-Jun phosphorylation, ultimately inducing apoptosis. Furthermore, ML324 treatment significantly inhibited tumor growth in bladder cancer patient-derived xenograft (PDX) models. Our findings reveal the presence of a Kdm4a-Sqle-ROS-JNK/c-Jun signaling axis that regulates oxidative stress balance, offering a novel strategy for targeted therapy in bladder cancer.

Abstract A052: RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction

Abstract RNF43_p.G659fsV*41 mutation (RNF43659mut) is found in approximately 8% of colorectal cancers (CRCs) and is enriched in microsatellite-instability high (MSI-high) tumors. Previously, we reported that RNF43659mut promotes tumor growth independent of WNT signaling and activates PI3K/AKT signaling in CRC, conferring vulnerability to PI3K/MTOR inhibition. In addition, RNF43659mut was associated with decreased interferon signaling suggesting a link with anti-tumor immunity. Thus, in the current work we investigated the role of RNF43659mut in CRC immunomodulation. The immune-cell landscape was evaluated in RNF43659mut compared with RNF43-wild type (wt) MSI-high CRCs in two cohorts; single-cell (sc)-RNA-sequencing data from treatment-naïve primary tumors from 34 patients (cohort-1) and multiplex immunofluorescence / whole exome sequencing data from an independent cohort of 91 patients (cohort-2). Healthy donor blood-derived natural killer (NK) cell responses and cytotoxicity were investigated in co-cultures with RNF43659mut isogenic tumor lines and with patient-derived organoids harboring RNF43659mut or overexpressing RNF43wt. A third patient cohort with MSI-high CRCs treated with immune checkpoint blockade was analyzed to understand the impact of RNF43659mut on immunotherapy efficacy. Interrogation of sc-RNASeq data in cohort-1 revealed a significant enrichment of NK cells in the RNF43659mut patient samples compared to the RNF43wt ones. This finding was also confirmed by multiplex immunofluorescence in cohort-2. Single-cell transcriptomic analyses in cohort-1 showed increased inhibitory markers on NK cells despite their enrichment in RNF43659mut tumors. RNAseq analysis of sorted NK cells co-cultured with RNF43659mut isogenic tumor lines further supported their dysfunctional state. Flow cytometric analyses of NK cell maturation, activation, and inhibitory markers in NK cells co-cultured with RNF43659mut isogenic tumor lines and organoids highlighted decreased levels of NCAM1 (CD56)dim and NCAM1 (CD56)bright subsets, FCGR3A (CD16), and KLRK1 (NKG2D), along with increased levels of KLRC1 (NKG2A), KIR3DL1 (NKB1) and TIGIT inhibitory markers. There was reduced NK cell mediated tumor cell killing in RNF43659mut isogenic tumor lines and organoids. We postulated that aberrant signaling of the PI3K/AKT pathway with RNF43659mut could lead to NK cell dysfunction. Indeed, siRNA-mediated blocking of PI3K/AKT pathway restored the NK-cell dysfunctional phenotype and tumor cell killing in co-cultures with RNF43659mut isogenic tumor lines and organoids. Conversely, overexpression of WT RNF43 in RNF43659mut organoids downregulated PI3K/AKT signaling and increased NK cell cytotoxicity. Lastly, MSI-high CRC tumors harboring RNF43659mut in patients treated with immunotherapy showed less benefit from immune checkpoint blockade. PI3K/AKT activation due to RNF43659mut in MSI-high CRC leads to NK cell dysfunction and reduced NK cell cytotoxicity. This could be responsible for immunotherapy failure, warranting further investigation. Citation Format: Pushpamali De Silva, Samantha Fitzgerald, Jules Cazaubiel, Matan Hofree, Tomotaka Ugai, Juha P Väyrynen, Dane Ford-Roshon, Lishan Fang, Ana Garrido-Castro, Nir Hacohen, Kimmie Ng, Shuji Ogino, Jonathan A Nowak, Marios Giannakis. RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A052.

Hepatocellular-Carcinoma-Derived Organoids: Innovation in Cancer Research.

Hepatocellular carcinomas (HCCs) are highly heterogeneous malignancies. They are characterized by a peculiar tumor microenvironment and dense vascularization. The importance of signaling between immune cells, endothelial cells, and tumor cells leads to the difficult recapitulation of a reliable in vitro HCC model using the conventional two-dimensional cell cultures. The advent of three-dimensional organoid tumor technology has revolutionized our understanding of the pathogenesis and progression of several malignancies by faithfully replicating the original cancer genomic, epigenomic, and microenvironmental landscape. Organoids more closely mimic the in vivo environment and cell interactions, replicating factors such as the spatial organization of cell surface receptors and gene expression, and will probably become an important tool in the choice of therapies and the evaluation of tumor response to treatments. This review aimed to describe the ongoing and potential applications of organoids as an in vitro model for the study of HCC development, its interaction with the host's immunity, the analysis of drug sensitivity tests, and the current limits in this field.

Oncofetal MCB1 Is a Functional Biomarker for HCC Personalized Therapy.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide and lacks biomarkers for personalized therapy. Herein, it is reported that MCB1 could be a novel oncofetal protein that is upregulated in the preneoplastic lesions and serum of early HCC patients. Functional studies reveal that MCB1 modulated p53 protein degradation to promote T-IC generation and drive HCC initiation. Furthermore, the MCB1/p53 axis is shown to determine the responses of hepatoma cells to conventional chemotherapeutics and predict transcatheter arterial chemoembolization (TACE) benefits in patients. Importantly, MCB1 can mediate sorafenib/lenvatinib resistance by downregulating two essential drug targets fibroblast growth factor receptor 1 (FGFR1) and vascular endothelial growth factor receptor 3 (VEGFR3) expression in a proteasome-dependent manner. Patient-derived tumor organoids (PDOs), patient-derived xenografts (PDXs), and patient cohorts analysis suggested that MCB1 levels in HCCs may determine the distinct responses to conventional therapeutics and targeted drugs. Furthermore, treatment of targeted drugs-resistant HCC with adeno-associated virus (AAV) targeting MCB1 or a proteasome inhibitor restores targeted drug response, suggesting their clinical significance in HCC combinational therapy. In conclusion, these findings demonstrate that MCB1 could act as a driver for HCC initiation, a contributor to drug resistance, and a biomarker for individualized HCC therapy.

BRCA promoter methylation in triple-negative breast cancer is preserved in xenograft models and represents a potential therapeutic marker for PARP inhibitors.

Most triple-negative breast cancers (TNBC) are sporadic in nature and often associated with dysfunction of the BRCA1 or BRCA2 genes. Since somatic BRCA mutations are rare in breast cancer (BC), this dysfunction frequently is the result of BRCA promoter methylation. Despite the phenotypic similarities of these tumors to those with germline or somatic BRCA mutation, the evidence of response to PARP inhibitors is unclear. We analyzed the prevalence of BRCA promoter methylation in 29 BC metastases through the well-established Illumina Infinium EPIC Human Methylation Bead Chip. In cases with BRCA methylation, the xenograft of the same tumor was tested. Additionally, we compared BC xenografts with an identified BRCA methylation to their matched primary tumors and subsequently investigated the efficacy of PARP inhibitors on tumor organoids from a BRCA2 promoter-methylated BC. BRCA2 promotor hypermethylation was identified in one pleural metastasis of a young patient as well as in the xenograft tissue. We also identified five more xenograft models with BRCA2 promotor hypermethylation. Analysis of one matched primary tumor confirmed the same BRCA2 methylation. PARP inhibitor treatment of tumor organoids derived from the BRCA2 methylated xenograft tumor tissue of the young patient showed a significant decline in cell viability, similar to organoids with somatic BRCA1 mutation, while having no effect on organoids with BRCA1 wildtype. BRCA promotor hypermethylation seems to be a rare event in metastatic BC but is preserved in subsequent xenograft models and might represent an attractive therapeutic marker for PARP inhibitors.

From Cancer to Immune Organoids: Innovative Preclinical Models to Dissect the Crosstalk between Cancer Cells and the Tumor Microenvironment

Genomic-oriented oncology has improved tumor classification, treatment options, and patient outcomes. However, genetic heterogeneity, tumor cell plasticity, and the ability of cancer cells to hijack the tumor microenvironment (TME) represent a major roadblock for cancer eradication. Recent biotechnological advances in organotypic cell cultures have revolutionized biomedical research, opening new avenues to explore the use of cancer organoids in functional precision oncology, especially when genomics alone is not a determinant. Here, we outline the potential and the limitations of tumor organoids in preclinical and translational studies with a particular focus on lung cancer pathogenesis, highlighting their relevance in predicting therapy response, evaluating treatment toxicity, and designing novel anticancer strategies. Furthermore, we describe innovative organotypic coculture systems to dissect the crosstalk with the TME and to test the efficacy of different immunotherapy approaches, including adoptive cell therapy. Finally, we discuss the potential clinical relevance of microfluidic mini-organ technology, capable of reproducing tumor vasculature and the dynamics of tumor initiation and progression, as well as immunomodulatory interactions among tumor organoids, cancer-associated fibroblasts (CAFs) and immune cells, paving the way for next-generation immune precision oncology.