3D Cell Culture Research Articles

Abstract 1705: 3D growth modulates the competition between STAT3 and STAT5 in breast cancer

Abstract Approximately 13% of women are diagnosed with invasive breast cancer. Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that is often inappropriately activated in breast cancer and is frequently associated with Triple Negative Breast Cancer (TNBC). STAT5 can also be activated and inappropriate activation of STAT5 alone or with STAT3 is generally correlated with a more favorable prognosis, a lower grade tumor, and a better response to therapies compared to inappropriate activation of STAT3 alone. Therefore, knowing the activation status of STAT5 and understanding how STAT5 activation can attenuate STAT3-driven breast cancers could help guide therapeutic choices and patient treatment. STAT3 and STAT5 can regulate a set of overlapping target genes. For example, STAT3 and STAT5 can regulate BCL6 expression, where STAT5 represses expression and STAT3 enhances it. Importantly, STAT5 outcompetes and preferentially binds in the presence of activated STAT3. This suggests that the overlapping target genes could play an important role in understanding how activation of STAT5 can lead to a more favorable breast cancer compared to activation of STAT3 alone. This previous work was conducted in a 2D cell culture model; however, 3D models can better mimic clinical settings and the use of 3D models can help bridge the gap. Therefore, understanding the relationship between STAT3 and STAT5 in 3D is important. SK-BR-3 cells (HER2+ cells with low basal STAT3 activity) and MDA-MB-231 cells (TNBC cells with constitutive STAT3 activity) are being compared between 2D cell culture and 3D media suspension. We have shown using chromatin immunoprecipitation that STAT3 DNA-binding is enhanced in 3D at certain binding sites, whereas STAT5 is not. STAT5 has reduced dominance and preferential binding over STAT3 for BCL6 in 3D compared to 2D. In other cases, STAT3 binding is decreased upon STAT5 activation in 3D but not 2D. To begin to understand these differences, we analyzed phosphorylation of these STATs in 2D and 3D. Phospho-STAT3 levels were similar between 2D and 3D in SK-BR-3 cells when activated with LIF stimulation but were higher in 3D in the MDA-MB-231 cells which contain constitutive STAT3 activity. STAT5 activation by prolactin stimulation did not directly affect STAT3 phosphorylation in 2D or 3D but does affect STAT3 binding in 3D growing cells. However, prolactin induced phospho-STAT5 seems to be reduced in 3D in both cell lines. We are currently analyzing additional binding sites to better define the differences between STAT3 and STAT5 mediated gene regulation in 2D and 3D since STAT phosphorylation does not fully address it. Overall, our data suggests that differences between 2D and 3D breast cancer models will be important for translation to patient tumors and understanding the relationship between STAT3 and STAT5 will help elucidate how STAT5 activation status can be used when treating patients with breast cancer. Citation Format: Alexandra Elizabeth Temple, Sarah R. Walker. 3D growth modulates the competition between STAT3 and STAT5 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1705.

Abstract 6045: Development and characterization of ATC-324: An AUTOTAC-based androgen receptor degrader for prostate cancer treatment

Abstract Prostate cancer (PCa) progression is largely driven by the androgen receptor (AR), making it a prime target for therapy. However, therapy resistance often arises due to AR mutations and splice variants, such as AR-v7. AUTOTAC (AUTOphagy-TArgeting Chimera) is a novel protein degradation platform that utilizes the autophagy-lysosomal pathway to selectively degrade disease-causing proteins. In this study, we characterize ATC-324, an AUTOTAC-based AR degrader designed for the treatment of PCa. ATC-324 comprises enzalutamide linked to YT 6-2, a small molecule activator of the autophagy receptor p62/SQSTM1. ATC-324 induces the formation of the AR/p62 complex while activating autophagic flux, leading to the autophagy-lysosomal degradation of AR. To characterize ATC-324, we employed various AR-dependent, AR-null, and enzalutamide-resistant PCa cell lines. Given that bone metastasis is a major clinical complication of castrate-resistant PCa associated with greater morbidity and mortality, we utilized the bone-in-culture array (BICA) assay, an ex vivo bones culture model, to investigate the impact of ATC-324 on PCa cell survival and growth in the bone microenvironment. We demonstrated that ATC-324 effectively degrades wild-type, mutant, and splice variants of AR. In addition, ATC-324 induces AR-dependent apoptosis and cytotoxicity. Importantly, ATC-324 maintains its potency in enzalutamide-resistant PCa cells. Utilizing the BICA assay, we showed that ATC-324 exhibits cytotoxicity against 22Rv1 bone micrometastases at even lower concentrations than in 2D cell culture. In conclusion, our study demonstrates the successful development and characterization of ATC-324 as a potent and selective degrader of various forms of AR. Furthermore, its effectiveness against enzalutamide-resistant cells indicates its capability to overcome therapy resistance. The observed cytotoxicity against bone micrometastases suggests a therapeutic potential for combating bone metastasis, a major complication of castration-resistant PCa. Our study underscores the potential of the AUTOTAC platform to selectively degrade disease-causing proteins Citation Format: Tri Pham, Tae Hyun Bae, Ki Woon Sung, Abdo Najy, Alaleh Zamiri, Hyejeong Jang, Su Ran Mun, Seongho Kim, Dongping Shi, Steven Kregel, Elizabeth Heath, Michael Cher, Yong Tae Kwon, Hyeong-Reh Kim. Development and characterization of ATC-324: An AUTOTAC-based androgen receptor degrader for prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6045.

Abstract 6000: A novel pathway controlling anticancer drug induced immunogenic cell death

Abstract Acting through estrogen receptor alpha (ERα), in orthotopic mouse xenograft models and a PDX, our non-competitive anticancer drug ErSO induces complete or near complete regression of primary and metastatic therapy-resistant ERα-positive breast, ovarian and endometrial tumors. ErSO is also highly effective in ovarian cancer PDOs from patient ascites. Unlike most anticancer agents which inhibit cancer cell proliferation or induce apoptosis, ErSO induces immune-cell-activating necrosis through sustained hyperactivation of the anticipatory unfolded protein response (a-UPR) pathway resulting in ATP depletion and cell swelling followed by membrane rupture. The medium from cancer cells killed by ErSO not only robustly activates mouse and human macrophages, but also dramatically increases monocyte migration. ErSO therefore has the potential to help extend the reach of immunotherapy to many solid tumors that do not express neoantigens. However, unlike apoptosis which has an established signaling pathway, how ErSO induces necrosis was unknown. From a genome-wide CRISPR-Cas9 screen in MCF-7 cells with negative selection against ErSO, we identified FGD3, a guanine exchange factor (GEF) of Rho GTPase Cdc42 as the top target. Consistent with the screen, through subsequent knockout and overexpression of FGD3 in human breast cancer cells, we found that the knockout cells have significant resistance to ErSO while the overexpressing cells exhibit increased sensitivity to killing by ErSO in both 2D cell culture and a 3D organoid model. Our data indicates that this protein is not an upstream regulator of the ErSO-induced necrosis pathway. Instead, it is a regulator of the last stage of necrosis - cell membrane rupture. Thus, we identified an actin reorganization signaling pathway with multiple components that plays a pivotal role in whether the breast cancer cell responds to a-UPR activation and swelling by membrane rupture and necrotic cell death or reorganizes its actin enabling the cancer cell to survive. RNA-seq and other studies indicate that by regulating actin reorganization, this axis is important in ErSO-persister breast cancer cells exhibiting long-term survival in ErSO. This work enables identification of breast cancer patients whose elevated levels of components of this signaling pathway make them most likely to benefit from this novel therapy. Notably, FGD3 knockout cells also show considerable resistance to necrosis or necroptosis induced by other anticancer drugs. Here, we describe a therapy that induces immunogenic cell death in ERα-positive breast, ovarian and endometrial cancer cells. We also show that, as cancer cells respond to agents that induce immunogenic cell death through membrane rupture, an actin regulating pathway plays a critical role in life-death decisions. Citation Format: Junyao Zhu, Santanu Ghosh, Darjan Duraki, Matthew Boudreau, Musarrat Jabeen, Chengjian Mao, Ben H. Park, Georgina Cheng, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. A novel pathway controlling anticancer drug induced immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6000.

Abstract 4226: Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions

Abstract The goal of the study was to determine the metabolic and morphological changes, via high Content Imaging, in 3D models of osteosarcoma cancer cells using human derived hydrogels with different protein composition, Obagel and Obagel ECM. Furthermore, we evaluated the relevance of the adipose tissue microenvironment in Adipose Derived Stem Cells (ADSC) linage and Tumoroids development. Methods: Adipose derived Stem cells were cultured in a modified 3D human and fat and bone linage differentiation as studied via histology and Immunofluorescence. Osteosarcoma spheroids were cultured in Obatala Sciences’ human-derived hydrogels ObaGel and ObaGelECM and control media. Organoid structure and phenotypic changes were analyzed via live cell imaging on the Incucyte S3 and Nikon high content imaging device. Then, adipocyte- cancer cell crosstalk was evaluated using pooled adipocytes co-cultured with KHOS spheroids. Adipose derived Stem cells were cultured in a modified 3D human derived hydrogel dictates that promotes the differentiation potential toward fat and adipose tissue simultaneously in microenvironments within the organoid. Furthermore, the results demonstrated that ObaGel and ObaGel-ECM 3D cultures can support the growth and proliferation of KHOS tumor spheres during an extended culture period. Metabolic changes and phenotypic changes associated to ObaGel and ObaGel-ECM differentially facilitated tumoroids growth and migratory behavior. Conclusions: Human derived hydrogels are developed to support 3D culture of Bone and Osteosarcoma cells to recapitulate the phenotypic changes associated with their metastatic potential. The use of human derived 3D culture systems can accelerate the understanding of the role of the microenvironment in bone development and the pathogenesis and progression of Cancers like Osteosarcoma as well as screening for drug development. Citation Format: Cecilia G. Sanchez, Arman Raz, Haley Lassiter, Trivia Frazier. Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4226.

Abstract 4227: Human-derived hydrogels to support phenotypic changes in ER+ breast cancer cell line

Abstract Adipose tissue plays a critical role in breast cancer incidence, progression, and response to therapy. The development of human derived 3D culture systems can accelerate the understanding of the role of environmental factors in the progression of Breast cancer as well as a model for preclinical studies during drug development. MCF-7 spheroids were cultured in Obatala Sciences’ Obavate and human-derived hydrogels ObaGel® and ObaGel®-ECM and control media. Organoid structure and phenotypic changes were analyzed via live cell imaging on the Incucyte S3. Migration studies were performed and gene expression analysis of EMT markers. Finally, adipocyte-breast cancer cell crosstalk was evaluated using pooled adipocytes co-cultured with MCF-7 spheroids. The results demonstrated that ObaGel® and ObaGel®-ECM 3D cultures support the growth and proliferation of MCF-7 tumorspheres during an extended culture period. In addition, MCF-7 cells exhibit the characteristic morphology of tumorsphere-forming cells when cultured in Obavate, with morphological changes in the ObaGel® and ObaGel®-ECM 3D. ObaGel® and ObaGel®-ECM differentially supports MCF-7 migratory behavior and phenotypic changes characteristic of EMT. Conclusions: Human derived hydrogels are developed to support 3D culture of breast cancer cells and to recapitulate the phenotypic changes associated with their metastatic potential. Furthermore, the use of a human derived 3D coculture system provides a platform for the understanding of adipose tissue-breast cancer cell interactions as it relates to breast cancer initiation, progression, and treatment response. Defining these interactions will support the development of a tool for future use in patient stratification and precision medicine in identifying underlying causes of breast cancer progression and response to treatments. Citation Format: Cecilia G. Sanchez, Katie Hamel, Emma Rogers, Jordan Robinson, Haley Lassiter, Trivia Frazier, Christopher Williams. Human-derived hydrogels to support phenotypic changes in ER+ breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4227.

Abstract 3167: Impact of gemcitabine modification on redox-driven responses in pancreatic cancer organoids cultured in distinct 3D microenvironments

Abstract Pancreatic ductal adenocarcinoma (PDAC) poses a formidable challenge in oncology, characterized by its high lethality and limited treatment options. The advanced stage at diagnosis, aggressive metastasis, and the constrained efficacy of conventional therapeutic drugs collectively contribute to the grim prognosis associated with PDAC. Gemcitabine, a nucleoside analog and the longstanding standard chemotherapy for pancreatic cancer, encounters a significant hurdle in its efficacy due to the development of resistance. To establish a cancer-acquired resistance model applicable to Gem-modified drugs, it is crucial to account for the dense extracellular matrix (ECM) composition characteristic of PDAC. In this investigation, we employ three-dimensional (3D) cell culture models, specifically utilizing pancreatic cancer patient-derived organoids (PDOs) within a 3D matrix with tunable density and stiffness by incorporating variations in transglutaminase crosslinking rates. PDAC cells from different patients exhibit diverse adaptations within these altered gel matrix environments, manifesting differences in morphology, doubling time, and responses to drugs. Notably, PDAC cells display pronounced stemness characteristics in the dense matrix, featuring a more rounded and compact appearance and an increase in ABC transporters.Addressing the challenge of gemcitabine resistance, modifications such as gemcitabine conjugates with stearate (Gem-S) have shown promise in preclinical studies. Exploring the effects of both Gem and Gem-S in a viscoelastic-specific ECM model derived from PDAC patient-derived organoids, distinct responses based on matrix stiffness come to light. The physical properties of the matrix exert a significant influence on cellular function, thereby impacting the effectiveness of therapeutic agents. Intriguingly, within a stiffer environment, Gem-S demonstrates higher sensitivity compared to Gem, suggesting the potential application of Gem-S in the dense pathological PDAC environment. Further investigation reveals that Gem-S treatment induces a noteworthy increase in reactive oxygen species (ROS) and HIF-1 expression compared to Gem. Surprisingly, the role of multidrug transporters in Gem-S sensitivity is limited, while the downregulation of NRF (Nrf2) after Gem-S treatment hints at distinct mechanisms underlying the development of drug resistance. This study provides valuable insights into the intricate interplay between gemcitabine resistance and the PDAC microenvironment. Understanding the matrix-specific responses opens avenues for tailored therapeutic interventions, offering the potential for improved outcomes in PDAC patients. Citation Format: Bo Han, Shuqing Zhao, Edward Agyare, Xueyou Zhu, Jose Trevino, Sherise Rogers, Enrique Velazquez, Jason Brant, Payam Eliahoo, Jonathan Barajas, Ba Xuan Hoang. Impact of gemcitabine modification on redox-driven responses in pancreatic cancer organoids cultured in distinct 3D microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3167.

Abstract 4258: An innovative PDX and patient-derived 3D platform for improved translatability of new medical entities

Abstract The preclinical platform landscape in oncology has radically changed in the last 15 years, mainly due to an increased ability to generate patient-derived models. Multiple 3D tumor cell cultures methods, either from established cell lines or from primary cultures, have been developed to assess the activity of therapeutic agents in vitro. These methods are strongly influenced by the experimental conditions, such as cell culture conditions, the presence of 3D supports like Matrigel, and the experimental design of the assay, such as the choice of the dose and the duration of the assay. The ability of these methods to predict drug efficacy in the patient relies not only in the optimization of the experimental settings, but also on the representativeness of the patient population, and on the prevention of model drifts due to clonal selection and/or tumor cell adaptation to culture conditions with passages. In this project, we aimed to develop an innovative 3D cell model platform that addresses all these experimental challenges. To do so, we took advantage of our low passage Tumorgraft platform, a bank of more than 1500 patient-derived xenografts (PDXs) well characterized at molecular and pharmacological level and highly representative of the patient population, to generate the Tumorgraft3D platform. We derived TumorGraft3D models from low passage PDXs, by using proprietary, indication-specific culture conditions. By keeping these models at low passage, we were able to avoid clonal drifts. Moreover, the use of indication specific media allowed the preservation of patient’s and PDX’s tumor histology, molecular traits at genetic, gene expression and proteomic level, and displayed proliferation rates that recapitulate those observed in the parental PDXs. So far, we have successfully developed over 100 models from breast, ovarian, prostate, NSCLC, colorectal and gastric indications, with success rates up to 90%. Ex-vivo testing of standard of care highlighted different response profiles in accordance with the molecular feature of these models. The tumor dissociation process and culture conditions optimized with PDXs were also used for the development of 3D cultures derived from patients’ samples. These data demonstrated that our 3D platform can integrate the complementarity of our PDX and patient-derived 3D platform, which lies in the possibility of evaluating test agent activity in the repeatable and reproducible setting provided by the patient-derived 3D models, as well as in models directly derived from patients with more heterogeneous and indication-specific establishment success rate, and throughput dependent on the amount of primary tumor material. Citation Format: Samaneh Kamali, Stefano Cairo, Fu-Ju Chou, Taylor Light, Mara Gilardi, Brandon Walling, Christine Baer, Hsiu-Wen Tsai, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. An innovative PDX and patient-derived 3D platform for improved translatability of new medical entities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4258.

Abstract 941: Patient-derived model systems of endometrial cancers for disease modeling and drug sensitivity testing

Abstract Background: Endometrial cancer (EC) is the most common gynecological cancer in the US, with increasing incidence and mortality rates. The survival rate lags behind four decades ago (81% vs 87%), underscoring the critical need for more effective treatment strategies. The heterogeneous nature of EC contributes to varied outcomes with current treatments. This project aims to establish reliable EC models for characterizing each individual tumor, distinguish optimal drug treatment, and determine specific drug effect mechanisms for personalized therapy. Methods: Freshly collected tumors were used for patient-derived xenograft models (PDXs) and patient-derived primary cancer cells (PDCs). Immunohistochemistry (IHC) staining was used to confirm the tumors grown in mice faithfully replicate the characteristics of patient tumor tissues. FDA-approved anticancer drugs were used to screen the optimal drugs to inhibit tumor cell proliferation and further utilized to inhibit tumor growth using PDXs. Results: We have collected 26 EC tumors and characterize them by DNA mutation and RNA-seq analysis. We implanted them in NSG mice, and 16 tumor samples have successfully grown in mice with a success rate of 61%. Most PDXs can be faithfully passaged to three generations. From the 16 PDXs, we created six novel PDCs. Using 2D and 3D spheroid cell culture, we screened 133 FDA-approved oncology drugs in the EC-PDC models. The drug screening results clearly highlighted several standout drugs, including CUDC-907 (a dual PI3K/HDAC inhibitor), two histone deacetylase (HDAC) inhibitors including romidepsin and panobinostat, four topoisomerase II (TOP II) inhibitors including mitoxantrone, daunorubicin, doxorubicin, and epirubicin, two proteasome inhibitors including carfilzomib and bortezomib, 2 DNA-directed RNA synthesis inhibitor dactinomycin and plicamycin, omacetaxine mepesuccinate (protein synthesis inhibitor), and valrubicin (DNA synthesis inhibitor). These drugs have demonstrated a significant capacity to inhibit tumor cell proliferation. Our pilot studies using single drugs, CUDC-907, romidepsin and mitoxantrone, achieved moderate drug effects. Combine mitoxantrone with romidepsin or CUDC-907 show surprisingly synergistic effects in multiple PDC models. The combination strategy will be tested in PDXs in the near future. Conclusion: Our unique PDXs and PDCs are excellent models for representing various characteristics of EC and testing novel therapeutics. This current study presents a promising direction for developing personalized therapy options for EC patients and provides a platform for further investigation of drug mechanisms and tumor development. Future studies will also involve etiology, such as chronic psychological stress, DNAm age and intratumoral microbiome. This information will help with endometrial cancer prevention, diagnosis, and prognosis. Citation Format: Tianyue Li, Xiaohao Huang, Riley Rosenmeyer, Samuel Robinson, Kazi Salsabil, Yiqin Xiong, Xiangbing Meng, Shujie Yang. Patient-derived model systems of endometrial cancers for disease modeling and drug sensitivity testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 941.

Abstract 210: Carcinogenic influence of e-cigarette aerosols on breast and lung cancer models

Abstract Background and Purpose: With the rising popularity of E-cigarettes among the youth, there is an urgent need to understand their long-term health implications, particularly the potential for carcinogenesis. Although tobacco smoking is established as a major cancer risk factor, the carcinogenic potential of E-cigarettes remains underexplored. Given that metastatic cancer accounts for most cancer-related deaths in the United States, elucidating the biological impacts of E-cigarette aerosols on cancer progression is critical for developing effective interventions. This study aims to address this critical gap by exploring the effects of E-cigarette aerosols on cellular models pertinent to breast and lung cancer. Methods: Mouse breast epithelial cells (HC11), triple-negative breast cancer cells (4T1), and human lung adenocarcinoma cells (Calu-3) were exposed to E-cigarette aerosol at the air-liquid interface for one hour, followed by a 24-hour recovery period. Results: Our study revealed that E-cigarette aerosols with mint flavor induce epithelial-to-mesenchymal transition (EMT) in both normal mammary epithelial and breast cancer cells. Furthermore, we also observed activation of the TGF pathway and an increased expression of stemness-related markers (OCT4, SOX2, NANOG, KLF4, and c-MYC) in 4T1 cells exposed to mint and menthol-flavored E-cigarette aerosols in both 2D and 3D cell culture environments. Microscopic examination at a 10X magnification level revealed that 4T1 and Calu-3 cells exposed to menthol-flavored E-cigarette aerosols exhibited signs of cellular disassociation and detachment whereas the control cells maintained discoidal aggregate morphology. Conclusion: The data presented here illuminate a potential link between E-cigarette use and carcinogenic processes, challenging the notion of E-cigarettes as a safe smoking alternative. This research is of paramount importance to cancer research as it unveils possible new avenues for tumor progression, emphasizing the necessity for a critical evaluation of E-cigarette products. Citation Format: Rizwana Begum, Shilpa Thota, Naveen Chintala, Biplov Sapkota, Abhishek Pandit, Joseph Francis. Carcinogenic influence of e-cigarette aerosols on breast and lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 210.

Abstract 6778: New microplate platform with cryopreserved 3D tumor spheroids for faster and convenient pre-clinical testing

Abstract Drug discovery and safety testing depend on predictive in vitro models, including multicellular tissues with organotypic functions. While traditional 2D tumor cell cultures have been extensively used in early drug screening due to their user-friendly nature, automation compatibility, and high throughput, they fall short in representing the intricate 3-dimensional structure, physiology, and complex multicellular nature of actual tumors. However, generating such 3D tissues for each test campaign is labor intensive. In addition, not all researchers have the resources to establish these 3D models. To pave the way, we developed a new platform that offers a range of cryopreserved 3D tumor models in micro plate format allowing for high-content screening. It enables users to start testing compounds within a 3-to-5 days simply by thawing an ARCTis™ plate (Always Ready Cryo Tissues) and following clear instructions. Another advantage of these cryopreserved 3D models derives from the fact that they are made in larger batches which means the start point for compound testing is always the same. This improves data quality, maximizes the test window length, and makes the data comparable across work groups. Currently, our growing portfolio consists of 25 tumor cell lines from ATCC representing the most common solid tumors. Their functionality has been determined based on efficacy tests for each cell line with the apoptotic agent Staurosporine. Various cell lines exhibited compact spheroids with a round morphology, less compact aggregates with non-spherical morphology, or loose cell-cell interaction aggregates, which can correlate with their tumor site of origin. Upon treatment with Staurosporine the dose-response profiles indicated growth inhibition, tumor shrinkage, or tumor cell killing effects based on size assessment and cell metabolic analysis (ATP). Moreover, we demonstrated the robust performance of these assays from plates stored for up to 6 months at -80°C.In conclusion, this groundbreaking approach addresses the limitations of traditional 2D cell cultures by providing a high-throughput, in-plate frozen, 3D cell culture platform. By offering a combination of a more physiologically relevant representation of tumors with a robust frozen-storage and time flexible assay platform our method holds great promise for advancing cancer drug discovery by easing the exploration of complex drug testing screens in vitro. Citation Format: Nils Goedecke, Simon Stroebel, Simon Hutter, Tala Issa, Irina Agarkova, Laure-Anne Ligeon, Olivier Frey. New microplate platform with cryopreserved 3D tumor spheroids for faster and convenient pre-clinical testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6778.

Abstract 3090: Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community

Abstract The NCI60 screen was established in the 1980s and introduced in the 1990s as a screening service for the worldwide cancer research community. The screen features 60 human tumor cell lines that represent 9 cancer types including non-small cell lung, colon, central nervous system, ovarian, renal, prostate and breast, as well as leukemia and melanoma. On a weekly basis, synthetic molecules, natural products, and biologics are screened against the 60-cell line panel to evaluate their anticancer potential. On average, more than 13,000 compounds are screened annually for investigators throughout the world. Cell growth and cytotoxicity were assessed by the sulforhodamine B (SRB) assay, performed in a 96-well format, that measures cellular protein content with an absorbance readout and enabled unprecedented throughput in the 1980s. Although the SRB assay supported a robust screening platform for three decades, its sensitivity and throughput are limited. Recently the NCI’s DCTD introduced the HTS384 NCI60 as a modernized screening platform. While the same 60 cell lines are evaluated, the HTS384 NCI60 is fully automated and performed in a 384-well format to enable a higher throughput. Additionally, the 48-h test agent exposure period of the SRB assay was increased to 72 h, which may result in lower 50% growth inhibition values (GI50) for some agents. A highly sensitive CellTiter-Glo luminescence readout for cell viability, based on cellular ATP content, was selected to enable comparisons of the NCI60 monolayer data to 3D cell culture models where the same assay chemistry can be applied. The HTS384 NCI60 was evaluated by screening a library of 1,003 U.S. Food and Drug Administration-approved and investigational oncology agents at five concentrations. Assay performance of the 60 cell lines was assessed from the controls of 30 - 60 microplates per cell line. The average doubling time of the 60 cell lines during the 72-h exposure period ranged between 19 h for NCI-H460 (17 - 23 h, n = 57) to 117 h for MDA-MB-468 (43 - 272 h, n = 60). The average signal-to-background ratio of all 60 cell lines was 89 (3,413 microplates) and ranged from 6 for OVCAR-5 (3 - 13, n = 54) to 229 for SF-539 (141 - 566, n = 60). The average coefficient of variation for microplate vehicle controls (n = 14) of all 60 cell lines (n = 3,413) was 5.2% and ranged from 3.2% for SK-MEL-28 (1.4 - 7.7%, n = 60) to 11.1% for HL-60(TB) (5.2 - 20.2%, n = 59). The average Z’-factor value of all 60 cell lines (n = 3,413) was 0.82 and ranged between 0.66 for HL-60(TB) (0.39 - 0.84, n = 59) to 0.89 for MALME-3M (0.79 - 0.95, n = 59), SF-295 (0.49 - 0.97, n = 59), SK-MEL-28 (0.74 - 0.95, n = 60), and UO-31 (0.8 - 0.94, n = 54). The development and performance of the HTS384 NCI60 screen will be described. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, Thomas S. Dexheimer, Eric M. Jones, Thomas Silvers, John R. Britt, Ronald C. Taylor, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3090.

Abstract 953: Integration of functional precision medicine assay for high grade glioma management: A single institution experience

Abstract Despite the advances in precision oncology, genomic approaches have not significantly improved outcomes for high grade gliomas (HGGs) as once was anticipated. Recurrent HGG pose even greater therapeutic challenge. The integration of functional precision medicine (FPM), in which drugs screens are performed on live tissue, into clinical routine practice presents a promising alternative by offering personalized treatments based on the unique characteristics of the patient’s tumor. Here, we present our institutional experience of integrating a spheroid-based drug screening assay (3D PredictTM Glioma) in the treatment management of HGG patients to evaluate its feasibility and efficacy as a therapeutic tool in a clinical setting. We also sought to determine factors that related to assay success. Tissue from intracranial lesions of patients with presumed HGG and planned surgical resection at our institution were collected for ex vivo 3D cell culture and challenged with 12 drug agents to determine patient-specific response parameters. The impact of potential variables, such as ki67, tumor pathology, and shipment timing, on the assay’s success was evaluated. Clinical correlation was established between ex vivo response and clinical response in HGG patients. 57 samples, including 24 upfront HGGs, 32 recurrent HGGs, and one medulloblastoma, were sent for spheroid formation and drug testing. In total, 57.9% (33/57) or 23% (13/57) of the assays were successful and resulted in complete or partial functional profiling data, respectively. We conducted a multivariable analysis of our cohort to determine which variables (i.e. tumor volume sent, ki-67 proliferation index, tumor percentage determined by DNA sequencing by Tempus Lab, recurrent status, sample shipment time) were predictive of assay success. The only variable significantly associated with assay success was tumor percentage with samples comprised of >70% tumor leading to assay success, 40%-70% leading to 60% chance of success, and <40% having 0% success. Among 46 patients with partial or complete assay success, 26 patients (56.5%) had a targetable mutation and 17/26 yielded a moderate or full response to one or more of their corresponding targeted drugs in the assay. Treatments were newly administered or altered from prior treatments in 40/57 patients (70%) based on FPM results. Our study demonstrates the technical feasibility of incorporating FPM approaches in the development of treatment regimens for patients with HGG. Success was correlated to tumor percentage present in the sample which indicates intraoperative communication between surgeon and pathologist can lead to improved assay results. Our institutional protocol demonstrates that Integrating FPM into routine clinical practice is possible and can serve as a valuable guide in the refinement of therapeutic recommendations for HGG patients. Citation Format: Grace Guzman, Sahana Bettadapura, William Jeremy Shelton-Correa, Taylor Brooks, Melissa Rayner, Christopher Wardell, Analiz Rodriguez. Integration of functional precision medicine assay for high grade glioma management: A single institution experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 953.

Abstract 6769: Applications of a novel fibrous extracellular matrix for advanced in vitro cancer models

Abstract Compared to traditional in vitro two-dimensional (2D) planar cell culture system, three-dimensional (3D) mammalian cell culture can better recapitulate physiological environments and the complexity of natural tissues, hence they are crucial in modern biomedical research. Extracellular matrices (ECM) play a key role in in vitro mammalian cell culture systems. The role of the ECM on in vitro 3D cell culture are: 1) to simulate an in vivo-like physiological environment, 2) support cell-cell and cell-ECM interactions, 3) promote cell adhesion, migration, and communication, 4) aid in tissue development, morphogenesis, differentiation, and functioning. Although basal membrane extracts (BME) are the most popular type of ECM supporting 3D modeling in vitro, they often have significant limitations including limited long-term stability, significant batch to batch heterogeneity, restricted cell movement, and relatively high cost for culturing many cancer cell lines. In this investigation, we employ a novel micro-fibrous scaffold that closely mimics natural ECM, providing porous and interconnected networks for 3D culturing of hepatic cells, lung cancer, breast cancer, pancreatic cancer. As revealed by optical and scanning electron microscopy, the scaffold promotes significant cell-to-cell and cell-to-matrix interactions. Moreover, we observed improved cell growth in 3D culture over longer periods than 2D culture systems for all cell types examined. We also evaluated the anticancer efficiency of different drugs including Lapatinib, Cisplatin, Docetaxel, Doxorubicin and observed that 2D cultured cells were more sensitive to drugs treatment in comparison to 3D. Furthermore, ECM was observed to support 3D growth and structure formation with cell pools of primary cells achieved from disassociated human tumors. Tumor organoids were able to form within seven days and ready for anticancer screening within 14 days. We achieved an 80% success rate in developing breast cancer organoids from patient tissues. Additionally, tissue granules were also able to be cultured under the same condition, which allowed us to investigate the invasion of cancer cells into tissue. The primary cells from human tumors formed cell clusters resembling the original tumor tissue's structure. In conclusion, the novel micro-fibrous scaffold, closely mimicking natural extracellular matrixes, promotes significant cell-to-cell and cell-to-matrix interactions, while also facilitating cell growth over extended periods providing a more physiologically relevant platform for evaluating drug responses for various cancer cell types, further showing the importance of 3D culture systems in cancer research. Citation Format: Navneet Kaur, Dazhi Yang, Jamel Ali. Applications of a novel fibrous extracellular matrix for advanced in vitro cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6769.

Abstract 512: Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles

Abstract Background: Traditional cell culture methods involve growing cells on flat surfaces, which do not fully replicate the human physiological conditions. In contrast, 3D cell cultures accurately reproduce aspects of the in-vitro malignant and non-malignant cell behavior. However, differences in tumor sensitivity platforms between 2D vs. 3D models might be relevant for some, but not for other drugs. Methods: The colorectal cancer cell line HCT-116 was cultured and plated in flat bottom well plates for the 2D condition and in Ultra Low Attachment round-bottom plates for the 3D condition. After plating, cells were treated with chemotherapies and targeted therapies for four days; cellular sensitivity was assessed through a viability assay. Four-parameter logistic (4PL) dose response curves were generated and IC50s were quantified to understand the differences in responses between 2D and 3D cellular environments. Results: HCT-116 exhibited similar responses to platinum drugs, taxanes, and several other tested drugs in both platforms, with the exception of gemcitabine (2D IC50 0.04 mcM; 3D IC50 12.8 mcM), lapatinib (2D IC50 23; 3D IC50 418), tucatinib (2D IC50 23.3; 3D IC50 79.3), and palbociclib (2D IC50 7.0; 3D IC50 24.0). Interestingly, these drugs share a common mechanism of action, which is the inhibition of cell cycle progression from G1 to S phase. Hence, we hypothesize that this shared mechanism may account for the observed differences in drug efficacy between the two environments. Conclusions: Similar dose responses have been observed with various drugs on both platforms, yet drugs that inhibit cell cycle progression from G1 to S phase, revealed higher sensitivity in 2D and more resistance in the 3D platform. Confirmatory cell-cycle results analyses are in progress. Further research is needed to understand if this applies to patient samples. Citation Format: Jahanvi Kumar, Chiara Maestri, Rajeshwar Nitiyanandan, Ivan Trus, Ricardo J. Parker, Brigitte Apfel, Christian Apfel. Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 512.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1566.

Abstract 6785: KIYA-PREDICT™ XPDX: An ex vivo 3D spheroid platform for modeling in vivo responses to multiple classes of oncology therapeutics

Abstract Patient-derived xenografts (PDX) are a powerful and commonly used tool in preclinical drug development as they represent an in vivo platform for screening drug response in human tumors that recapitulate many molecular and histological features of the original patient tumor. However, these models are costly and time-intensive, necessitating the development of ex vivo platforms for assessing drug response to PDX-derived tumors to de-risk in vivo studies by aiding in model selection and reducing the number of animals and costs associated with in vivo experimentation. The KIYA-PREDICT™ platform is a clinically validated, ex vivo 3D cell culture platform with demonstrated ability to predict patient response to chemotherapy in high grade glioma and ovarian cancer. Here, we apply this platform to profiling XPDX from XenoSTART’s comprehensive library of well-characterized models as a tool for assessing drug response and mechanism of action ex vivo while also aiding in model selection for in vivo studies. Over 100 XPDX models have been profiled ex vivo for response to several classes of cancer therapeutics, including chemotherapy, targeted therapy, and antibody-drug conjugates (ADCs), demonstrating robust concordance with in vivo drug response. CDK4/6 inhibitors palbociclib and ribociclib evaluated ex vivo in breast XPDX display strong correlation to in vivo response and recapitulate palbociclib resistance. Similarly, G12C inhibitors sotorasib (AMG-510) and adagrasib (MRTX849) screened against a panel of non-small cell lung cancer XPDX ex vivo match in vivo response and drug resistance. Finally, ex vivo and in vivo responses to FDA approved ADC enfortumab vedotin were evaluated in a panel of bladder cancer PDX, with ex vivo and in vivo responses showing good concordance and expected modulation based on Nectin-4 expression levels. The combination of the KIYA-PREDICT™ platform with XenoSTART’s XPDX catalogue is a flexible and powerful tool that can be utilized for streamlining preclinical drug development for a wide range of therapeutic classes. Citation Format: Aaron L. Carlson, Natalie W. Dance, Kimberly J. Burgess, Danielle Y. Nadeau, Melissa Millard, Alyssa Simonson, Armando Diaz, Michael Wick, Teresa M. DesRochers. KIYA-PREDICT™ XPDX: An ex vivo 3D spheroid platform for modeling in vivo responses to multiple classes of oncology therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6785.

Abstract 240: Mix and match - A comprehensive pipeline for matched patient-derived PDX and PD3D® modelsfor cancer research and preclinical drug development

Abstract Relevant in vivo and in vitro cancer models have been invaluable for our current understanding of cancer biology, as well as for therapy development. Using appropriate models in the drug development process significantly supports reliable and swift decision making in proceeding along the value chain. In parallel, animal experiments can be designed more specific to the scientific and regulatory needs, resulting in shorter development cycles and eventually the availability of better therapies for cancer patients. Patient-derived xenografts (PDX) are generated by implantation of cancerous tissue from a patient’s tumor either under the skin (ectopic) or into the organ of tumor origin (orthotopic) of an immunocompromised mouse and are the most used in vivo model for preclinical drug development. Patient-derived 3D cell culture models (PD3D®) are gaining increasing significance as relevant in vitro cancer models that recapitulate the original tumor tissue’s biology and are suitable for high-throughput drug screening. The combination of both platform’s increases the translational relevance of the development pipeline and offers the possibility to enlarge the respective panels of tumor models. Tackling an unmet medical need, we initially created matched mesothelioma PD3D® and PDX models. Mesothelioma is an aggressive cancer with poor prognosis and limited treatment options. Development of new therapies is hampered by shortage of available relevant tumor models. We created PD3D® models from PDX mesothelioma models and vice versa and generated drug sensitivity profiles with standard of care such as Gemcitabine, platinum derivates and a number of targeted agents to confirm their suitability as matched models for future projects. We were able to prove that models express stable histological, phenotypic features and drug sensitivity profiles across the two platforms. With this proof-of-concept study, the feasibility to combine the PD3D® biobank comprising more than 500 models from CELLphenomics with the PDX bank from CRL including 700 PDX models, has gained momentum. In consideration of the changing regulatory landscape, the combination of in vitro and in vivo platforms is a crucial step towards a drug development pipeline that incorporates the principles of 3R and increases its translational value due to fully human technologies being the corner stones of the drug development process. Citation Format: Jürgen Loskutov, Eva Oswald, Lena Wedeken, Christoph J. Reinhard, Julia Schueler, Christian R. Regenbrecht. Mix and match - A comprehensive pipeline for matched patient-derived PDX and PD3D® modelsfor cancer research and preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 240.

Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

Hydrogel-based 3D cell cultures can recapitulate (patho)physiological phenomena ex vivo. However, due to their complex multifactorial regulation, adapting these tissue and disease models for high-throughput screening workflows remains challenging. In this study, a new precision culture scaling (PCS-X) methodology combines statistical techniques (design of experiment and multiple linear regression) with automated, parallelized experiments and analyses to customize hydrogel-based vasculogenesis cultures using human umbilical vein endothelial cells and retinal microvascular endothelial cells. Variations of cell density, growth factor supplementation, and media composition are systematically explored to induce vasculogenesis in endothelial mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes in 384-well plate formats. The developed cultures are shown to respond to vasculogenesis inhibitors in a compound- and dose-dependent manner, demonstrating the scope and power of PCS-X in creating parallelized tissue and disease models for drug discovery and individualized therapies.

Abstract 6834: Periostin involvement in cellular senescence in ovarian cancer

Abstract Purpose: Despite improvements in care and advancements in the understanding of ovarian cancer (OC) pathobiology and genetics, survival rates remain disappointingly low compared to other types of cancer. This is due to a lack of diagnosis strategies, high recurrence rates, and the development of chemo-resistant diseases, which require synergy between cancer cells and the tumor microenvironment (TME). We have demonstrated the functional importance of a TME gene, periostin (POSTN), in OC and recurrent OC, using in vitro and in vivo models. Our objectives were to identify differentially expressed genes by POSTN expression in the TME that contribute to the progression of ovarian cancers. Procedures: Gene expression data was generated from an ovarian cancer cell line, HEYA8, cultured in a high POSTN conditioned 3D cell culture environment, using the NovaSeq 6000 system to generate RNA-seq data. The data was analyzed to determine the genes expressed at different levels between the HEYA8 cells grown with or without POSTN-high conditioned medium (CM). The gene functional annotation and KEGG pathway enrichment were further analyzed from 10,115 significant differentially expressed genes between the HEYA8 cells grown in high levels of POSTN (CMPOSTNhigh) or low levels of POSTN (CMCTL) culture conditions, using DAVID bioinformatics. Results: Using a cell culture and co-culture model, we found that OC cells cultured under conditioned media containing CMPOSTNhigh exhibited faster cell migration, more invasiveness (p=0.006), more chemo-resistance (p<0.05), and increased resistance to paclitaxel-induced apoptosis, compared to OC cells cultured with control medium (CMCTL). OC cell lines cultured with CMPOSTNhigh showed increased side population cells relative to CMCTL -cultured cells, suggesting POSTN plays important roles in cancer stem cell enrichment. POSTN-transfected 3T3-L1 cells exhibited more intracellular and extracellular lipids and were linked to increased cancer cell expression of the oncogene fatty acid synthetase. In correspondence to the functions of POSTN in OC cells, the pathways identified to be significantly impacted by POSTN expression included, but were not limited to, the cell cycle, apoptosis, p53 signaling, MAPK signaling, and PI3K-Akt signaling pathways. Interestingly, we also found cellular senescence pathways was one of the most significantly impacted by POSTN suggesting that POSTN may play roles in ovarian cancer via the regulation of cellular senescence, which has not yet been explored. Conclusions: These data emphasize the importance of TME factors in tumor progression and prognosis via various ways in ovarian cancer, establishing promising potential therapeutic targets of OC. Citation Format: Jihua Feng, Ethan Nguyen, Jocelyn Reyes, Zhiqing Huang. Periostin involvement in cellular senescence in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6834.

Abstract 2047: Scale up and scale down approaches for screening of 3D patient-derived cell models

Abstract 3D cell culture is becoming more prevalent as scientists work to better model human physiology using in vitro systems. However, differences in handling 3D cell cultures compared to traditional 2D cell lines have limited adoption of 3D techniques in compound and cell therapy screening experiments. To facilitate use of patient-derived 3D tumoroid models, we developed protocols for both scale up and scale down of these models in Gibco™ OncoPro™ Tumoroid Culture Medium. OncoPro Tumoroid Culture Medium is a serum-free, conditioned medium-free culture medium designed to promote the growth of patient-derived 3D cancer models (tumoroids, cancer organoids). OncoPro medium is compatible with a suspension culture approach for easier handling compared to traditional embedded culture formats. We studied the growth of tumoroids in different flask sizes during suspension culture to optimize scale up for downstream assays. Tumoroid growth rates and morphologies were consistent across non-tissue culture treated flask sizes, and viable cell yield increased with surface area during scale up. In some cases, as many as 100 × 106 dissociated tumoroid cells can be recovered from a single Nunc™ TripleFlask™ after a week of growth. After scaling up, tumoroids were dissociated and seeded in multiwell plates to test multiple screening conditions (compound identity, compound concentration) in parallel. Dissociated cells were seeded in 96-well plates using both manual and automated liquid handling (Tecan Fluent 780). Due to the tendency of 3D cell models to aggregate and fall out of solution, initial seeding in a 96-well flat bottom plate resulted in high well-to-well variability. Optimization of cell seeding protocols led to standard deviations in tumoroid metabolic activity, number, and size that were comparable to manual seeding, with a coefficient of variation of less than 3% between wells for multiple tumoroid lines when automation was implemented. Generally, variability in cell seeding was lower when using the optimized, automated cell seeding protocol compared to manual methods. After seeding, cells were grown for three days to form tumoroids prior to the addition of compounds. At this time point, there was low well-to-well variability in tumoroid metabolic activity (CV<2%). Compounds and reagents to analyze cell health could also be added using automated liquid handling prior to analysis on plate readers or high-content imaging platforms. Citation Format: Colin Paul, Anthony Chatman, Amber Bullock, Xiaoyu Jenny Yang, Garrett Wong, Brittany Balhouse, Chris Yankaskas, Erik Willems, Matt Dallas, David Kuninger. Scale up and scale down approaches for screening of 3D patient-derived cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2047.