Advances in breast cancer organoid for individualized treatment

1895P - Breast cancer organoids model treatment response of HER2 targeted therapy in HER2-mutant breast cancer

BackgroundMammary Paget’s disease (MPD) is an uncommon cutaneous intraepithelial malignancy with ulceration of the nipple or areola. Its pathogenesis and genomic mutation remain largely unknown and no cell lines are established from primary tumors.MethodsWe collected surgical tumor specimens from a 65-year-old Chinese woman diagnosed with MPD and established patient-derived breast cancer (BC) organoids from MPD using organoid culture technology.ResultsWe successfully propagated BC organoids from a patient with MPD for more than 6 months. The organoids were cultured for long-term expansion without any change in spherical organoid morphology. Besides, the spherical organoid morphology did not change when they underwent cryopreservation after resuscitation. The H&E staining and immunohistochemistry analyses showed the similar morphological and histological features of the organoids compared with their paired original BC tissues. The organoids retained positive expression of breast cancer biomarkers: estrogen receptor, progesterone receptor, antigen Ki-67 and negative expression of human epidermal growth factor receptor 2. We also showed that MPD organoids recapitulated the unique genomic landscape including copy number alterations, mutational load, mutational signatures and cancer gene mutations by whole genome sequencing. In situ senescence-associated acid beta galactosidase assay confirmed senescence phenomenon existed in the process of organoids culture and there was no significant difference in the proportion of senescent organoids after organoid passage and resuscitation.ConclusionsOur results suggested that an effective platform for ex vivo BC organoids from MPD patients could be used to explore clinicopathological and genomic characteristics of these patients.

Background: In recent years, the incidence of breast cancer (BC) is high and on the rise. Organoids, as a new in vitro 3D culture model, play a huge role in BC treatment and drug development. Objective: To summarize the domestic and foreign research experience in BC organoids in drug screening, in order to provide new ideas for BC treatment.Summary: This article briefly summarized the mechanism of action of commonly used drugs for BC, including trastuzumab, tamoxifen, olaparib, paclitaxel, pembrolizumab, and the effects of HER-2 drugs and CDK4/6 inhibitors on BC organoids, clarified the importance of BC organoids, and drew the conclusion that more accurate treatment could be achieved by using this model. The successful establishment of papillary carcinoma organoids further demonstrated the feasibility of cultured 3D models in vitro. On this basis, drug sensitivity tests for different drugs were conducted, and it was concluded that fluvestrant had the best anticancer effect, thus proving that BC organoids played an important role in drug screening. In addition, organoid technology also has a good effect in the treatment of Triple-negative breast cancer (TNBC). Using this model for large-scale drug detection, four compounds including papirinestat, patinib, JIB-04 and TAK-901 showed effective activity on TNBC organoids, indicating that organoids can promote drug discovery. It is concluded that BC organoids are a potential therapeutic model, and the development of this technology provides an innovative approach to treating BC.

e12558 Background: Breast cancer is one of the most malignant diseases threatening the health of our society. Triple negative breast cancer (TNBC) is an aggressive histological subtype representing 15–20% of all breast cancers. Comparing with other breast cancer subtypes, TNBC is characterized with limited treatment options and a worse clinical outcome. Resistance to chemotherapies is common for TNBC. Therefore, continuously developing new anti-cancer drugs is of great importance for TNBC. Nature products a source of medicinal leads, but lacking suitable pre-clinical models, which could faithfully reflect in vivo tumor property, prevents these compounds from further characterizing. Now cancer organoids, as a new in vitro 3-D culture technique, were demonstrated to largely retain the biological characteristics of tumors from patients, and potentially served as an ideal platform for drug sensitivity test. Methods: Here we set up system to efficiently establish breast cancer organoids from clinical patients and further test the sensitivity of these organoids to various nature products. Results: A total of 29 TNBC organoid models were established with fresh biopsy or surgery tumor tissues from TNBC patients. Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in TNBC PDOs were evaluated with qPCR, western blot, Immunohistochemistry (IHC). A high-throughput cell viability assay was established for identifying lead compounds from a unique collection of 969 natural products. Primary hits were serial diluted and further evaluated for IC50 and percentage of inhibition. Preliminary mechanism of action (MOA) studies was performed with gene reporter assays, western blot and qPCR. Interestingly, organoids derived from paclitaxel resistant TNBC patients also show less sensitivity to paclitaxel in vitro, which indicated a high clinical relevance. Conclusions: Triple negative breast cancer organoid, as a good pre-clinical model largely containing tumor in vivo property, provides an ideal platform for new drug discovery and inhibitor screening. Moreover, serving as in vitro replacements for breast cancer, organoid models make it possible to test drug sensitivity for individual patient to achieve precise and personalize medication.

Breast cancer organoid microarray technology, as an emerging research tool, has been used to construct breast cancer organoid models with physiological functions in vitro by simulating the tumor microenvironment, providing powerful support for in-depth understanding of breast cancer pathogenesis, drug screening, personalized treatment, and prognosis assessment. The aim of this study is to delve into the research hotspots and prospective directions of breast cancer organoid microarrays, and to better understand the opportunities in the research hotspot areas. The Web of Science Core Collection database was used to search for articles published on the combination of organoid and microarray research in breast cancer, using bibliometric methods to analyze authors, research institutions, countries, journals, references, and visualized by CiteSpace and VOSviewer. A total of 151 documents were included in this study, revealing a steady annual increase in publications with country, China publishing the most articles with the highest citation (1667). Fudan University, Sun Yat-Sen University, and China Medical University contributed the most research with five articles published. Nanyang Technological University and the Massachusetts Institute of Technology are the institutions with the highest connection strength, indicating that their cooperation and communication in this field play an important role. Over the past decade, the focus of breast cancer research has shifted from the optimization and standardization of organoid culture models to the simulation of the tumor microenvironment and the establishment of disease models. Subsequent research trends include improving the pathological representativeness of the models, drug screening and personalized therapy. This study provides an objective and comprehensive analytical perspective on the research prospects of breast cancer organoids, pointing out the way for future research.

Background Patient-derived cancer organoids, which reliably conserve original features of tumors, are emerging as an excellent model for predicting therapy response and drug screening. Developing optimized 3D high-throughput drug screening platform to establish patient-derived cancer organoids and simultaneously perform drug screening is essential for personalized medicine. Methods We established normal breast organoids (n=4) and breast cancer organoids (n=10) from 20 fresh surgical specimen (normal 7, tumor 13 cases). A number (500-2000) of normal and cancer cells were automatically dispensed with the ASFA™ Spotter ST and organoids were generated by hydrogel hanging-drop culture on Cellvitro™ Pillar platform (Medical & Bio Decision, South Korea). Organoids were subjected to drug screening for 17 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 5 days. Drug cytotoxic effect was assessed by calcein AM staining. Acquisition and analysis of high-content 3D organoid images were peformed using ASFA™ SCANNER (Medical & Bio Decision, South Korea). The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. We analyzed a drug response index (DRI) using a prediction alogorithm to evalute drug sensitivity (DRI<-0.5) and resistance (DRI>0.5). Results We summarized the DRI value of patient-derived breast organoids of 7 drugs (Table 1). 6 tumor organoids (2T, 6T, 8T, 10T, 13T, 15T) showed high sensitivity to Docetaxel, Doxorubicin, Paclitaxel, and Gemcitabin while 4 tumor organoids (1T, 9T, 14T, 16T) were less sensitive and resistant. 2 tumor organoids (10T, 16T) were sensitive to Tamoxifen and 2 tumor organoids (6T, 8T) show high sensitivity to palbociclib and erlotinib. Normal organoids show less sensitivity and resistance to chemotherapeutic drugs. Drug response index >0.5 : resistancy top 30%, Drug response index <-0.5 : sensitivity top 30%. Conclusions Herein, we developed the hydrogel hanging-drop culture on Cellvitro™ Pillar platform for easily and rapidly high-throughput drug screening in patient-derived organoids using a small number of cells by testing clinically actionable drugs at different concentrations. There were different drug response indeces for each individual organoids to chemoreagents and targeted drugs. We anticipate that 3D high-throughput drug screenings platform based on patient-derived organoids can provide the information to predict drug response and allow for finding more appropriate therapy for individual patients. Table 1.DRI values of patient-derived breast organoids of 7 drugs.Patient No.Tumor/NormalDocetaxelPaclitaxelDoxorubicinTamoxifenGemcitabinePalbociclibErlotinib1TTumor1.051.261.080.060.590.26-2TTumor-0.64-0.72-0.60-0.08-0.79--6TTumor-0.98-0.99-2.330.00-1.44-2.31-1.608TTumor-0.32-0.50-0.250.64-1.07-1.47-0.669TTumor1.050.980.610.870.430.610.4410TTumor-0.84-0.77-0.78-1.680.790.610.0913TTumor-0.87-0.430.560.70-0.510.611.1714TTumor0.861.261.180.481.81--15TTumor-1.59-1.45-1.41--0.71-0.13-16TTumor1.051.261.18-1.70-1.22-2NNormal1.051.260.650.741.810.610.7810NNormal1.05-0.910.810.870.880.610.084NNormal-0.31-0.25-0.530.25-0.190.61-1.575NNormal1.051.130.380.75-0.630.610.10 Citation Format: Jungeun Kim, Hoe Suk Kim, Ga Yeon Kim, Kyung hyeun Park, Seung yeon Ryu, Sangeun Lee, Dong Woo Lee, Bosung Ku, Han-Byoel Lee, Wonshik Han. Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer 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 P5-02-02.

Background: Breast cancer is one of the most malignant diseases threatening the health of our society. It is estimated that there will be more than 300,000 new cases of breast cancer in the US in 2018. Currently, surgery combined with chemotherapy is the most common strategy for breast cancer treatment. However, due to the cancer heterogeneity induced drug resistance, continuously developing new anti-cancer drugs is of great importance. Nature compounds extracted from traditional Chinese herbs were demonstrated to have anti-cancer activity through cell based experiments, but lacking suitable pre-clinical models, which could faithfully reflect in vivo tumor property, prevents these compounds from further characterizing. Now cancer organoids, as a new in vitro 3-D culture technique, were demonstrated to largely retain the biological characteristics of tumors from patients, and potentially served as an ideal platform for drug sensitivity test. Breast cancer organoids study was relatively limited when comparing with other type of cancer such as colon cancer. In this study, we set up system to efficiently establish breast cancer organoids from clinical patients and further test the sensitivity of these organoids to various nature compounds. Methods: We collected fresh breast cancer tissues from 17 patients after surgery and established organoid models. Different dosage of herb derived nature compounds such as Gypenoside, Berberine, Oxymatdne, Sophoridine, Betaine, Chelerythrine Chloride, Harmine, Cantharidin were used to treat each organoid clone. Luminescent cell viability assay was used to indicate tumor survival rate. Results: Organoid were successfully established for all 17 patients. A Short Tandem Repeat (STR) analysis was employed to detect sample contamination and HE(hematoxylin and eosin) stain was used to confirm the histological features. All organoids were resistant or slightly response to Oxymatdne, Sophoridine, Gypenosides, Betaine. For Berberine and Cantharidin, 10 out of 17 and 14 out of 17 organoids showed dose-dependent inhibition efficacy respectively, and the rest showed resistance. For Chelerythrine Chloride and Harmine, only organoids from specific patient showed sensitivity in a dose-dependent manner. Interestingly, different patients had distinct response pattern to these nature compounds, indicating organoids were useful in the anti-cancer lead screening and biomarker identification. Conclusion:Breast cancer organoid, as a wonderful pre-clinical model largely containing tumor in vivo property, provides an ideal platform for new drug discovery and inhibitor screening. Moreover, serving as in vitro replacements for breast cancer, organoid models make it possible to test drug sensitivity for individual patient to achieve precise and personalize medication. Citation Format: Zhang B, Xu X, Cai H, Sun Z. Anticancer potential evaluation of natural products with breast cancer organoids [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-04-03.

Highlights from Recent Cancer Literature

The molecular mechanisms of telomerase reverse transcriptase (TERT) upregulation in breast cancer (BC) are complex. We compared genetic variability within TERT and telomere length with the clinical data of patients with BC. Additionally, we assessed the expression of the TERT, MYC, TP53 and SP1 genes in BC patients and in BC organoids (3D cell cultures obtained from breast cancer tissues). We observed the same correlation in the blood of BC patients and in BC organoids between the expression of TERT and TP53. Only in BC patients was a correlation found between the expression of the TERT and MYC genes and between TP53 and MYC. We found associations between TERT genotypes (rs2735940 and rs10069690) and TP53 expression and telomere length. BC patients with the TT genotype rs2735940 have a shorter telomere length, but patients with A allele rs10069690 have a longer telomere length. BC patients with a short allele VNTR-MNS16A showed higher expression of the SP1 and had a longer telomere. Our results bring new insight into the regulation of TERT, MYC, TP53 and SP1 gene expression related to TERT genetic variability and telomere length. Our study also showed for the first time a similar relationship in the expression of the above genes in BC patients and in BC organoids. These findings suggest that TERT genetic variability, expression and telomere length might be useful biomarkers for BC, but their prognostic value may vary depending on the clinical parameters of BC patients and tumor aggressiveness.

2004P - Breast cancer organoids: A new tool for the prediction of drug penetration and patient outcome

Breast cancer is the malignant tumor with the highest incidence in women. Nowadays, the objects in vitro of models of this disease are mainly from breast cancer cell lines and patient-derived patient-derived xenograft (PDX). However, there is a significant gap between traditional cell lines and breast cancer solid tumors, meanwhiles, PDX is not highly consistent with patients due to different species. As a techonlogy, obtaining patient-derived tumor cells, combined with three-dimensional culture technology, adding cytokines that promotes the proliferation of breast cancer stem cells and inhibit their apoptosis, breast cancer organoids form a structure in vitro which is similar to tumor in the body. This model can not only study the occurrence and envolution of breast cancer, but is more prominent in clinical application. screening drugs by high-throughput, personalized treatment, textingtoxicity and immunotherapy. This article will review the breast cancer organoids, in evolution, source, culture system and clinical applications.

BackgroundBreast cancer is the most common malignancy among women globally. Organoid technology has emerged as a pivotal tool in breast cancer research due to its advantages in modeling tumor heterogeneity and the microenvironment. Despite rapid advancements in this field, a systematic bibliometric analysis to delineate research trends and challenges is lacking. This study aimed to analyze the research landscape, hotspots, and future directions in the field of breast cancer organoids from 2005 to 2024.MethodsPublications related to breast cancer organoids published between January 2005 and March 2024 were retrieved from the Web of Science Core Collection. Bibliometric tools (CiteSpace and VOSviewer) were employed to analyze collaboration networks (countries/institutions), author contributions, keyword co-occurrence clusters, and burst keywords.ResultsOver the past two decades, the annual publication output on breast cancer organoids has shown continuous growth. The 1618 included English publications garnered a total of 7,323 citations, with a mean citation count of 35.20 per article. The United States (n=666) and China (n=257) contributed over 50% of the publications. Harvard University was the most productive institution. Mina J. Bissell authored the highest number of publications (n=17). High-frequency keywords centered on personalized therapy, immunotherapy, and 3D bioprinting. Burst keyword analysis identified “gene expression” and “signaling pathways” as emerging trends (2019–2024). Key research hotspots include the application of patient-derived organoids (PDOs) for drug screening, co-culture modeling of the tumor microenvironment (TME) with immune components, and the integration of 3D bioprinting technologies.ConclusionThis study represents the first comprehensive bibliometric analysis to elucidate the evolution and research hotspots in breast cancer organoid research in recent years. The findings provide a thorough summary of the major achievements, persistent challenges, and future frontiers within this rapidly advancing field.

e13125 Background: Marketed methods are available for in vitro organoid growth for the purpose of translational science, evaluation of drug efficacy and patient treatment predictivity, but require needle biopsy of the tumor which is invasive to the patient. Non-invasive methods exist for isolating circulating tumor cells (CTC) from blood but require large volumes due to their low frequency (<1 in 105-106 PBMCs). Low CTC recovery limits the number of downstream analyses. To overcome these obstacles, Cellentia is introducing a novel in vitro system called R3CE (Rapid, Reproducible, Rare Cell Expansion), developed by AcroCyte Therapeutics Inc. (Taiwan), to the US market. The advantage of R3CE is that blood is used to generate viable representative cells of the patient’s tumor which can be expanded and banked, thus the number of characterization assays is flexible. The R3CE method allows for extensive drug screening within a rapid timeframe to inform patient treatment. This poster will show drug screening results on organoids derived from blood of breast cancer patients, and R3CE method transfer to Cellentia. Methods: For patient drug screening, 0.5 mL of peripheral blood was drawn from stage IV breast cancer patients. Cells were seeded onto proprietary plates, cultured for several days, passaged and screened for drug resistance or susceptibility to Cisplatin, Paclitaxel, 5-FU, Vinorelbine, and Gemcitabine by assessing the relative viability from luminescent measurements of drug treated organoids normalized to a mock control. For R3CE method transfer, frozen breast cancer organoids were thawed and seeded onto proprietary plates, grown for several days and used in the drug screen as described above. Results: Blood from five breast cancer patients (Patients A-E) were grown into organoids and used for screening drugs listed (Table). Relative viabilities were calculated. A threshold was set at 10% where relative viabilities above or below 10% showed the organoids were resistant (R) or susceptible (S) to killing by the drug candidate, respectively. The results of the R3CE drug test were compared to the clinical (Clin) outcome for each patient (clinical outcome is unknown (U) in some instances) and shown (Table). The drug test and the clinical outcome showed 100% concordance. Cellentia executed drug screening with the same drug panel utilizing frozen breast cancer organoids in the R3CE platform and demonstrated susceptibility in the drug screening. Conclusions: R3CE platform showed promising results as a personalized drug screening tool for breast cancer patients. Frozen breast cancer organoids for drug screening demonstrated successful method transfer to Cellentia. [Table: see text]

Background: Breast cancer is the second most common cancer in women, accounting for 15.3% new cancer cases and 6.7% cancer death in 2018. Chemotherapy, target therapy and endocrine therapy are main strategies for breast cancer treatment after surgery to prevent cancer recurrence and metastasis. The choice of these strategies is mainly based on the molecular and pathological markers as well as experience from clinicians. However, due to the heterogeneity of intra-tumor and also between different patients, it is hard for clinicians to make the best therapeutic regimen to target all the cancer sub-population at current stage. Developing more precise and personalized chemotherapy strategy for individual patient would be of great importance. Cancer organoids, established based on in vitro 3-D culture techniques, were demonstrated to largely retain the biological characteristics of tumors from patients, and made it possible to assess the responses to various strategies for individual patient. Comparing with other cancer type such as colon cancer, breast cancer organoids study was relatively immature. This study was designed to efficiently establish breast cancer organoids from clinical patients and further test the sensitivity of these organoids to therapeutic agents. Methods: Fresh breast cancer tissues from patients were dissected into small pieces and subjected to enzyme digestion to prepare a single cell suspension. Organoids were established from the single cell suspension in Matrigel and modified PDTO culture medium. Organoid clones derived from various cancer cells were heterogeneous, which were isolated with pipette tips to establish breast cancer organoids subclones. First line chemotherapy drugs such as Docetaxel, Adriamycin, and Fluorouracil, target therapy agents and endocrine therapy drug such as PD-991, Neratinib and Afimoxifene were used to treat each organoid clones. Cell survival rate was measured by CellTiter-Glo Kit and drug sensitivity was assessed by IC50 value. Results: 23 organoid subclones from 5 patients were successfully established. Drug response (effect) varied to individual organoid subclones from the same patient. Some subclones showed totally resistance while others were sensitive or parcial sensitive to indicated therapeutic agents, indicating the heterogeneity of breast cancer and different intra-tumor subpopulation have distinct drug response. The heterogeneity, especially the resistant subclones put forward to explain the acquired drug resistance and relapse in clinical practice. Conclusion:Breast cancer organoids are good in vitro models for drug sensitivity screening, which would assist clinicians make better chemotherapy strategy to achieve precise and personalized medication for patients. Moreover, organoid models would also be an ideal platform to help dissect the underlying mechanism of cancer heterogeneity induced drug resistance. Citation Format: Zhang B, Xu X, Cai H, Sun Z. Evaluation of breast cancer intratumor heterogeneity and its implications to the therapeutic agents with organoids subclones [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-04-02.

Introduction: Nicotinamide (NAM), a water-soluble form of niacin (Vitamin B3), is a precursor of nicotinamide-adenine dinucleotide (NAD+) and regulates cellular energy metabolism. In our previous study with metabolic RNA panel assay, we confirmed ALDOA (Aldolase A) is suppressed after treatment of nicotinamide in breast cancer organoids. ALDOA is a crucial glycolytic enzyme that turns fructose-1,6-bisphosphate (FBP) into glyceraldehyde-3-phosphate. Several studies have shown that high expression of ALDOA is associated with poor prognosis in various types of cancer. However, its function in breast cancer remains unclear. Methods: ALDOA expression in breast cancer was analyzed with METABRIC data set. A total of 79 fresh cancer tissues and tumor microarrays (TMA) with 402 breast cancer patients were enrolled. Metabolic RNA sequencing panel assay was performed with human breast cancer organoids after treatment of nicotinamide. The breast cancer cell lines including MCF7, T47D, and BT474 were generated to establish stable cells in which ALDOA expression is continuously reduced using lentiviral system. Total RNA was isolated from human breast cancer cells and performed RT-PCR for Sanger sequencing. Proliferation assay was performed using cell counting kit-8. We used transwell migration and invasion assay. To identify cancer stemness, we performed sphere formation assay. Mitochondrial function was measured using the Seahorse XF24 Flux Analyzer. Results: In our previous study, after the treatment of nicotinamide, ALDOA expression in breast cancer organoids was suppressed. So we assumed that ALDOA has an oncogenic function in cellular metabolism. The expression of ALDOA was much higher in breast cancer clinical tissues compared with normal tissues. High expression of ALDOA was correlated with the worse prognosis of breast cancer patients in METABRIC public data and our own cohort. Sanger sequencing in the cell lines showed no mutation in exon5 to exon9 which is known as mutation hotspot in breast cancer. Cell proliferation activity and ability of cellular movement were inhibited in ALDOA-downregulated breast cancer cells. In the sphere formation assay, we found that the tumor cells with low expression of ALDOA decreased cancer stem cell properties. When POU2F1 or POU2F2 was suppressed, ALDOA protein expression was also inhibited. ECAR and OCR concerning mitochondrial metabolic functions were also downregulated in the tumor cells with ALDOA suppression. Conclusions: In conclusion, we suggest that ALDOA might be a key biomarker inducing tumor aggressiveness by modulating metabolic processes in breast cancer. Our results suggest that nicotinamide can be a novel therapeutic candidate targeting in breast cancer with high expression of ALDOA. Citation Format: Da Sol Kim, Cheng Hyun Lee, Soo Young Park, Han Suk Ryu. New metabolic biomarker of ALDOA induces tumor aggressiveness in breast cancer and nicotinamide as a potential anti-tumor agent targeting ALDOA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 269.