MASEA: A microfluidic system for in situ evaluation of tumor angiogenesis in PDO-endothelial co-culture.

Advanced pancreatic cancer has a dismal prognosis and current treatment options (FOLFIRINOX, Gemcitabine/nab-paclitaxel [GnP]) are associated with toxicity. Although some patients achieve partial responses, most progress rapidly and become chemorefractory. While RNA subtypes, genomic alterations, and protein biomarkers have prognostic value, predictive biomarkers to guide therapy are needed. Patient-derived organoids (PDOs) are an increasingly popular model for predicting patient responses to standard-of-care therapy and investigating personalized therapy options. We present a novel biobank of 42 PDOs and drug profiling data with 5 standard of care agents and 3 kinase inhibitors. Tissue was processed from n=103 biopsies from 97 patients with a confirmed pathologic diagnosis of advanced (Stage III-IV) pancreatic ductal adenocarcinoma who presented to a single Canadian tertiary care centre between 2017-2020. Matched WGS was available in all cases. Our PDO generation success was 42/103 (41%). We observed a trend towards decreased establishment in tumors that were KRAS WT, TP53 WT, or had higher HRDetect scores. Conversely, polyploidy, SMAD4 WT, and major imbalances in mutant KRAS were associated with successful PDO establishment. These associations were not statistically significant after multiple comparisons correction, but suggest selection for success with more aggressive tumors. Drug profiling was performed on all 42 PDOs with the individual agents of FOLFIRINOX (5-FU, irinotecan, oxaliplatin), GnP (gemcitabine, paclitaxel), and three targeted agents (afatinib, trametinib, and talazoparib). Combination testing was also performed for gemcitabine + paclitaxel. Drug responses were measured through both viability and growth rate (GRMetrics). We found that GRMetrics minimized effects from different PDO growth rates. Matched clinical data were available for 23 patients who received FOLFIRINOX, 11 patients who received GnP, and one patient who received gemcitabine monotherapy. Similar to previous studies, we found that in vitro PDO responses to 5-FU, irinotecan, and GnP were correlated with patient responses based on RECIST criteria. Interestingly, and similar to previous reports in colorectal cancer PDOs, we found that oxaliplatin responses were not predictive of RECIST response. As expected, PDOs were resistant to afatinib (EGFRi), which reflects negative clinical trials, and may also be masked by use of EGF in growth media. A range of responses to trametinib (MEKi) were seen but were not correlated with KRAS allelic dosage. A similar range of response was seen to talazoparib (PARPi), but did not correlate with oxaliplatin response or HRDetect scores. In summary, we have established a novel biobank of PDOs from advanced pancreatic cancer patients. Notably, PDOs were less likely to establish from tumors that were KRAS WT or HR-deficient, even though these patients are likely to benefit from targeted approaches. Further investigation is required to develop PDO use in clinical drug prediction and drug discovery. Citation Format: Irene Y. Xie, Laura Tamblyn, Karen Ng, Eugenia Flores-Figueroa, Julie M. Wilson, Gun Ho Jang, Amy X. Zhang, Stephanie Ramotar, Anna Dodd, Nikolina Radulovich, Jennifer J. Knox, Grainne M. O'Kane, Steven Gallinger, Faiyaz Notta. Establishment of a novel living biobank of patient-derived pancreatic cancer organoids with genomic and drug response characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-077.

Background. Organoids have been widely accepted as 3D tumor models for patient-specific drug response. However, research involving non-small cell lung cancer (NSCLC) organoids is hampered by the lack of appropriate platforms. Although dome cultures show promising results, the relatively large size variability of patient-derived organoids (PDOs) hinders their application in drug response studies. Here, we report a microfluidic chip for maintaining high uniformity and requiring miniscule sample volume for PDO drug screening. Methods. The microfluidic chip includes a bottom microwell layer and a top microchannel layer. Each chip has 5 channels for parallel treatment conditions, with 30 U-shaped microwells (400μm dia x 250μm deep) per channel. PDOs were loaded from side channels and trapped by filters adjacent to microwells. NSCLC PDOs F231/F671 (NCI PDMR) were cultured and treated by KRASG12C inhibitor adagrasib (0-2000nM, 72h). PDO drug response was assessed via viability using fluorescent dyes. Results. On-chip PDO size after one-week incubation exhibited the coefficient of variation (CV) of 28% for F231 and 36% for F671, which was notably lower than measurements obtained using Matrigel dome (126%, n=3) and low attachment plate (80%, n=4). The F231/F671 showed viability >81% after 1wk (n=20). On-chip cytotoxicity results showed that both KRASG12C PDOs exhibited sensitivity to adagrasib, matching the clinical response, with IC50 values of 830 nM (F231) and 1324nM (F671). When cultured with fibroblast (WI-38) supernatant, the cytotoxic effect was mitigated due to the fibroblast-induced resistance, with IC50 increasing ≥2 ×. No statistical difference was observed for the fibroblast effect in the open-format microwells and our microfluidic chip (69.6±16.9% to 80.2±9.0% vs. 69.0±12.2% to 82.2±4.1%, respectively, following 500nM adagrasib for 72h), further underscoring the reliability of our chip for PDOs’ drug response studies. Application of continuous perfusion at 70nL/min yielded no statistical difference in viability from static conditions. Conclusions. Our microfluidic chip offers uniform PDO growth with perfusion allowing drug screening in almost real time. The on-chip response to adagrasib was clinically relevant. Resistance due to tumor microenvironment (TME) can be readily assessed using fibroblast supernatant, and could be expanded to other TME components in future studies to investigate their impact on treatment resistance. Citation Format: Qiyue Luan, Ines Pulido, Angelique Isagirre, Jian Zhou, Takeshi Shimamura, Ian Papautsky. Microfluidic chip for drug response studies in non-small cell lung carcinoma patient-derived organoids [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 234.

The coculture of patient-derived tumor organoids (PDOs) and autologous immune cells has been considered as a useful ex vivo surrogate of in vivo tumor-immune environment. However, the immune interactions between PDOs and autologous immune cells, including immune-mediated killing behaviors and immune-related cytokine variations, have yet to be quantitatively evaluated. This study presents a microfluidic chip for quantifying interactions between PDOs and autologous immune cells (IOI-Chip). A baffle-well structure is designed to ensure efficient trapping, long-term coculturing, and in situ fluorescent observation of a limited amount of precious PDOS and autologous immune cells, while a microbeads-based immunofluorescence assay is designed to simultaneously quantify multiple kinds of immune-related cytokines in situ. The PDO apoptosis and 2 main immune-related cytokines, TNF-α and IFN-γ, are simultaneously quantified using samples from a lung cancer patient. This study provides, for the first time, a capability to quantify interactions between PDOs and autologous immune cells at 2 levels, the immune-mediated killing behavior, and multiple immune-related cytokines, laying the technical foundation of ex vivo assessment of patient immune response.

Abstract Body: Characteristics of the PDAC tumor microenvironment (TME) such as desmoplasia and an immunosuppressive landscape contribute to its resistance to chemotherapy and immunotherapy approaches. To recapitulate and dissect the in vivo PDAC TME, we bioengineered a PDAC organoid-on-a-chip model by integrating patient-derived PDAC organoids on a microfluidic-based organ-on-a-chip. While the patient-derived organoids (PDOs) maintain the characteristics of the in vivo counterparts with high fidelity, the organ-on-a-chip provides a controllable and reproducible environment with stromal and immune niche cells, creating an in vitro model that may providing insights into PDAC drug resistance and allow testing for new therapies. The multicellular organoid-on-a-chip model is composed of 1) a central PDAC niche including patient-derived PDAC organoids, patient-derived cancer-associated macrophages (CAFs), tumor-associated macrophages (TAMs) and blood vessels, and 2) surrounding vascular networks for nutrient transport and drug delivery. We reproduced the hypovasculariy of PDAC stromal niche on the chip and found a lower level of CD31 expression in the PDAC organoid niche than in the normal pancreatic organoid niche (p<0.0001). Comparing with the niche without PDOs, the PDAC niche on chip demonstrated a higher expression level of α-smooth muscle actin (α-SMA) on CAFs (fold-change=2.32, p<0.0001) and a more extensive deposition of ECM components (e.g., collagen I, collagen III, collagen IV and hyaluronic acid, fold-change>=2.25, p⇐0.0002), indicating the existence of desmoplasia on chip. Profiling of cytokine levels in the media revealed that, compared to models used with normal pancreatic epithelial cells with the other cellular components, the PDAC organoid TME induced the upregulation of a series of pro-tumor cytokines including MIP-3α, IL-8, CXCL5, IL-13 (fold-change>=5.02), the inflammation-related cytokines MIF, IL-5, MCP-1 (fold-change>=1.82), and CAF-derived cytokines like CXCL10, HGF, CXCL1, CXCL12 (fold-change>=1.82). The immunosuppressive cytokine TGF-β was increased after PDOs were loaded on the chip (762.3 pg/mL vs. 222.6 pg/mL in conditioned medium), as was the proinflammatory cytokine IL-6 (1089.5 pg/mL vs. 81.3 pg/mL), was maintained at a higher level than that in the niche with normal organoids (380.5 pg/mL vs. 60.4 pg/mL at day 13). We also found that PDOs induced TAMs inclined to be M2-like (CD68+CD163+) phenotype on chip. In turn niche cells (TAMs and CAFs) contributed to the immunosuppression in PDAC TME: the secretion of cytokines like IL-6, IL-13, MIP-1β and HGF were downregulated without niche cells (fold-change⇐0.646). In conclusion, we have developed a PDAC organoid-on-a-chip recapitulates the PDAC TME and is promising to serving as a platform to study mechanisms of drug resistance in PDAC as well as test novel therapies. Citation Format: Lunan Liu, Diane M. Simeone, Weiqiang Chen. An organoids-on-a-chip model to recapitulate and dissect the tumor microenvironment of PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B057.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy driven by KRAS mutations in ~90% of cases, with high heterogeneity and limited efficacy of single targeted agents. Patient-derived organoids (PDOs) and xenografts (PDXs) offer promising platforms for personalized therapy by replicating tumor characteristics.MethodsWe established a PDAC biobank with 69% PDO and 31% PDX success rates from 66 patient samples. Next-generation sequencing (NGS) of 425 oncogenes was performed, followed by 32-drugs in vitro screening in PDOs. The synergistic effects of the MEK inhibitor trametinib combined with the mTOR inhibitor AZD8055 or the pan-CDK inhibitor flavopiridol were evaluated in PDOs and validated in matched PDXs. We also validated PDOs in predicting clinical gemcitabine/paclitaxel (Gem/PTX) responses.ResultsPDOs preserved tumor histological and genetic feature, with consistent drug responses across early and late passages. Trametini/AZD8055 exerted robust synergistic antitumor effects in all tested PDO and PDX models, while trametinib/flavopiridol failed in PDO/PDX-099. The Gem/PTX regimen achieved 75 -95% growth inhibition in PDOs, and the in vitro results were highly consistent with the in vivo efficacy in PDXs and the clinical CA19-9 remission of patients.DiscussionThis study effectively integrated two preclinical models, PDOs and PDXs, both in vitro and in vivo, which are highly regarded in the fields of drug discovery and personalized medicine. The trametinib/AZD8055 combination is a promising precision therapeutic strategy, and PDOs can serve as a reliable tool to guide clinical therapy selection. Despite limitations such as small sample size, lack of tumor microenvironment and immune components in the model system, this work provides important preclinical evidence for the clinical translation of PDOs in the personalized therapy of PDAC.

Patient-Derived Ovarian Cancer Organoids Mimic Clinical Response and Exhibit Heterogeneous Inter- and Intrapatient Drug Responses

Patient Derived Organoids (PDOs) are described as miniature, three dimensional (3D) cell cultures prepared from patient’s cancer cell to compare therapeutic responses in the laboratory and clinical settings. Personalized cancer medicine is a novel therapeutic strategy to identify a right treatment for the right patient in accordance with tumor’s genetic signature. Tumor Organoid models have innumerable benefits over pre-existing models which makes them a tremendous candidate in personalized cancer therapy as they mimic the physiology of the original tumor. Recently several studies have shown the value of these “tumor in-dish” approaches in personalized cancer medicine for preclinical drug screening and prediction of patient treatment outcome. The objective of this “Journal of Carcinogenesis and Mutagenesis” research topic is to advance our current understanding about PDOs as an attractive in vitro model system for studying tumor evolution and response to drugs and treatments.

Patient-derived organoids (PDOs) are emerging as preclinical models with promising values in personalized cancer therapy. The purpose of this study was to establish a living biobank of PDOs from patients with non-small cell lung cancer (NSCLC) and to study the responses of PDOs to drugs. PDOs derived from NSCLC were cultured in vitro, and then treated with natural compounds including chelerythrine chloride, cantharidin, harmine, berberine and betaine with series of concentrations (0.5-30 μM) for drug screening. Phenotypic features and treatment responses of established PDOs were reported. Cell lines (H1299, H460 and H1650) were used for drug screening. We successfully established a living NSCLC organoids biobank of 10 patients, which showed similar pathological features with primary tumors. Nine of the 10 patients showed mutations in EGFR. Natural compounds chelerythrine chloride, cantharidin and harmine showed anticancer activity on PDOs and cell lines. There was no significant difference in the 95% confidence interval (CI) for the IC50 value of chelerythrine chloride between PDOs (1.56-2.88 μM) and cell lines (1.45-3.73 μM, p>0.05). PDOs were sensitive to berberine (95% CI, 0.092-1.55 μM), whereas cell lines showed a resistance (95% CI, 46.57-2275 μM, p<0.0001). PDOs had a higher IC50 value of cantharidin, and a lower IC50 value of harmine than cell lines (p<0.05, 7.50-10.45 μM and 4.27-6.50 μM in PDOs, 3.07-4.44 μM and 4.69-544.99 μM in cell lines, respectively). Both PDOs and cell lines were resistant to betaine. Chelerythrine chloride showed the highest inhibitory effect in both models. Our study established a living biobank of PDOs from NSCLC patients, which might be used for high-throughput drug screening and for promising personalized therapy design.

Cancer remains the leading cause of mortality in the world, despite several cutting-edge technologies and established therapeutic regimens for cancer treatment. Therefore, the key to developing accurate and effective therapeutics is having a comprehensive knowledge of these complex molecular events. Patient-derived organoids (PDOs) represent a perfect model for studying cancer drug resistance and therapy. These cancer organoid models are cheaper alternatives to xenograft models and traditional two-dimensional (2D) cell culture model systems. All cancer organoid models are developed using iPSC-derived spheroids and tumor cells from different sources, which are then processed on a matrigel scaffold to get cancer organoids. The major advantage of these model systems is that they can recapitulate many functional and genetic characteristics of the same tumor tissues “in vitro”. These cancer organoids can be passaged, frozen, and preserved for further high-throughput screening analysis. PDOs are powerful tools for evaluating mutational profiles and testing cancer drugs for personalized therapy. Cancer organoids can also be used to study tumor microenvironment cell types by co-culturing the required cell types involved in the process of transformation, which allows us to study tumor microenvironment and tissue-tissue interactions in the tumor development and metastasis process. This leads to more accurate predictions of the process of tumor development and evaluation of responses of cancer drug-resistance in a particular patient to develop personalized therapies for cancer. However, several limitations to these cancer organoid models must be addressed and resolved to get a perfect system for cancer drug evaluation. Several scientists are working on it by developing standardized protocols and reagents to generate individual tissue organoids. It is hoped that major developments in technologies, such as organoids-on-chips, 3D bio-printing, and advanced imaging techniques, will improve the handling of these organoids more precisely. Further CRISPR-Cas9-based gene editing technology allows us to bioengineer normal organoids by introducing any combination of cancer gene alterations to derive cancer organoids. In this review, we focused on the development and improvement of various normal and cancer organoids for targeted tissues such as the lung, breast, colon, liver, and kidney and their use as model systems for cancer drug discovery and personalized therapy. We have also highlighted some of the uses of the latest technologies, such as microfluidics chips and 3D bioprinting, for deriving better cancer organoids-based in vitro models for future research on cancer therapeutics.

3101 Background: PDO is a promising translational tool that recapitulates the biology and drug response of donor cancer patient. However, an unmet need is to have PDO drug-screening data available for treatment decision making in clinic. We conducted a pilot study to determine whether PDO testing results will be available at critical treatment decision points in metastatic GI cancer patients. Methods: Metastatic GI cancer patients undergoing core-needle biopsy were eligible. Tumor cells isolated from ≤4 fresh biopsy tissues were grown in a Matrigel-based culture. PDO response to anti-cancer drugs were evaluated; and when available, correlated with donors’ clinical response to the same agent(s). PDO response was defined as IC50 &lt; 0.1 × published Cmax of the drug clinically; stable as IC50 between 0.1 to 10 × Cmax. Radiographic response was per RECIST criteria. Results: We enrolled 27 refractory metastatic GI cancer patients (9 colorectal [CRC], 9 pancreas, and 9 biliary tract). Median lines of therapy were 4, 2, and 2; the success rate of organoid establishment was 89%, 44%, and 55%, respectively. The median time from biopsy to availability of drug-testing data was 64 days (range: 24 to 93 days). The median time from biopsy to next CT re-staging in donors was 64 days. The established PDOs shared histological and genomic features with donor clinical tissue. PDO and clinical responses to the same agent(s) were correlated in 2 CRC donors including (1) BRAFV600E-mutated PDO responded to vemurafenib + panitumumab, as did the donor who had partial response (PDO drug-testing data were available 55 days post-biopsy, 23 days prior to restaging scan); (2) KRAS/FGF-dual amplified PDO had stable disease status to regorafenib, as did the biopsied lesion from the donor (73 days post-biopsy, 5 days post-scan). Conclusions: We showed the feasibility of completing PDO drug sensitivity testing in metastatic GI cancer patients within a short time that could impact clinical decision making, particularly in CRC. PDO drug response showed correlation with clinical response. With further refinement, PDO can be a powerful tool for personalizing cancer therapy in metastatic GI cancer patients.

e15169 Background: PDX and PDO are two of the most frequently applied avatar model systems used to predict treatment response to anti-cancer therapies. Despite their frequent use, and the significant financial and ethical costs associated with developing these models, there has never been a systematic assessment of their ability to predict matched-patient treatment response. We sought to define and compare the efficacy of PDX and PDO in predicting matched-patient response to treatment. Methods: We performed a systematic review and meta-analysis in accordance with PRISMA guidelines. MEDLINE and EMBASE were queried. Inclusion criteria: PDX or PDO derived from adult solid cancer patients treated with identical systemic anti-cancer agents as the matched patient, with response assessment performed for both patient and model. Fisher’s exact test and Kaplan-Meir estimator with log-rank test were used for statistical comparisons. A 6 criteria quality assessment method based on Newcastle-Ottawa scale was applied to patient-model pairs. Results: 21565 abstracts were screened. 274 were eligible for data extraction, with 411 patient-model pairs included (N = 267 PDX, N = 144 PDO). The most common cancer types were colorectal (N = 102, 25%) and ovarian cancers (N = 77, 19%). Most common treatment modalities were chemotherapy (244, 59%) and targeted therapy (122, 30%). 55% of models were responsive to therapy (N = 227). Overall concordance in treatment response between patient and matched models was 70%, with no difference between PDX and PDO ( Table ). No significant differences in sensitivity, specificity, positive- and negative predictive value (PPV and NVP) were observed (Table). 196 pairs (48%) and significantly more PDX had high quality data reporting (56% of PDX vs 33% of PDO, P &lt; 0.001). Of pairs with high quality data reporting, PDX had higher sensitivity, while PDO had higher specificity ( Table ). Patients whose matched PDO responded to therapy had longer median progression-free survival (mPFS; responders: 11.3 vs non-responders: 3.4 months P &lt; 0.01). For PDX this only remained true for high quality data pairs (mPFS; responders: 9.5 vs non-responders: 6 months P &lt; 0.01). Conclusions: This is the first study to systematically assess the utility of PDX and PDO as patient avatars. Together, these results suggest that PDO generally perform similarly to PDX as predictors of matched-patient response despite a lower ethical and financial burden. Performance metrics for PDX and PDO as predictors of matched-patient treatment response. All PDX (N = 267) PDO (N = 144) P-Value Concordance (%) 71 69 0.9 Sensitivity (%) 87 85 0.81 Specificity (%) 58 60 0.69 PPV (%) 64 52 0.1 NPV (%) 84 89 0.37 High Quality Data PDX (N = 149) PDO (N = 47) P-Value Concordance (%) 72 81 0.57 Sensitivity (%) 95 73 &lt;0.01 Specificity (%) 54 88 &lt;0.01 PPV (%) 61 84 0.07 NPV (%) 94 79 0.07

This PhD thesis explored the use of patient-derived organoids (PDOs) obtained from tumor tissue, to address research limitations and therapeutic challenges in pancreatic ductal adenocarcinoma (PDAC), characterized by a remarkable inter- and intra-tumoral heterogeneity, by an immunosuppressive and immune evasive microenvironment and resistance to conventional cytostatic drugs and immunotherapy. Two separate but complementary in vitro studies were conducted applying immune cell co-cultures based on PDAC PDOs to innovate PDAC research at the preclinical level and provide a platform to monitor therapeutic approaches. The first research priority outlines cell live imaging with OrganoIDNet, a platform for real-time characterization and monitoring of PDAC organoid response to both chemotherapy and immunotherapy. PDAC organoids, which closely mimic in vivo tumor behavior, were cultured either alone or in co-culture with immune cells, allowing assessment over time of not only cytostatic but also immunotherapeutic effects in individual patient-derived PDAC organoids. Initially, OrganoIDNet, a deep-learning-based algorithm capable of analyzing bright-field images of murine and human PDAC organoids acquired with live-cell imaging was established to evaluate the effects of gemcitabine, a nucleoside analog considered the gold standard in systemic chemotherapy. This study reveals distinct responses to gemcitabine in mouse and human PDAC organoids, emphasizing the platform sensitivity to minor variations. Real-time monitoring provided organoid morphological parameters such as eccentricity, in response to gemcitabine. Using OrganoIDNet, organoid healthy status was assessed using pixel intensity analysis, which classifies organoids as healthy or unhealthy based on brightness values. While gemcitabine-induced reduction in organoid count and area are significant, pixel intensity analysis depicts size-dependent effects of drugs on organoids and distinguishes between healthy and unhealthy status, both parameters undetectable by conventional endpoint viability assays, underlying OrganoIDNet's superiority in analyzing organoid behavior, such as size-dependent therapy effects over time. To account for organoid size heterogeneity, OrganoIDNet is capable of categorizing PDAC PDOs into five size bins, revealing size-dependent responses to gemcitabine. Smaller organoids increase in number, while larger ones decrease in response to treatment, highlighting the platform's capacity to identify size-based effects in organoid cultures in response to therapy. OrganoIDNet's accuracy in depicting anti-tumor efficacy was validated through the endpoint assay CellTiter-Glo, confirming its reliability in assessing organoid viability. Most importantly, the platform's potential to improve our understanding of personalized drug responses is underlined by the dynamic insights into organoid responses that cannot be captured by conventional endpoint assays. In addition, a new organoids/PBMCs sandwich-based co-culture protocol enabled longitudinal analysis of organoid responses to immunotherapy with the PD-L1 inhibitor Atezolizumab. The use of OrganoIDNet and imaging of live cells revealed an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added. The second part focused on investigating the potential of Mesovac, a mesothelin (MSLN)-based nanovaccine formulation alone or in combination with chemotherapy or the PD-L1 antibody Atezolizumab as an immunotherapeutic strategy for PDAC. This was achieved in multiple experimental stages, including in vitro T-cell stimulation assays, ex vivo T-cell expansion of reactive T cells, and immune cell co-culture experiments based on PDAC PDOs and stimulated T-cells. First, to assess Mesovac ability to induce an immune response, in vitro T-cell-based peptide stimulation was conducted using unfractionated peripheral blood mononuclear cells (PBMCs) from healthy donors. Although modest, an increase in IFN-γ+ T cells suggests the Mesovac potential to activate T-cell responses in vitro. To overcome variability and expand Mesovac-stimulated T cells, artificial antigen-presenting cells (aAPCs) were employed. These aAPCs, coupled with MSLN4 and adjuvants, enhance the reactivity of PDAC patient-derived T cells when exposed to Mesovac components. Further investigations explore the effect of Mesothelin-peptide-stimulated T cells on PDO growth and proliferation. The combination of Mesothelin-peptide-stimulated T cells with gemcitabine shows promising results in attenuating PDO expansion, particularly in synergistic effect with gemcitabine. Additionally, Mesothelin-peptide-stimulated T cells combined with Atezolizumab and FOLFIRINOX results in an increased efficacy in targeting PDAC organoids. Mesothelin-peptide-stimulated T cells treatment reduces the overall tumor cell population in PDAC PDO co-cultures as well as cells expressing PDAC-associated tumor markers, measured by flow cytometry. Moreover, cells expressing cancer stem markers, such as CD24, EpCAM or CD133, show an overall reduction upon treatment with FOLFIRINOX. This effect is even more evident with the addition of Atezolizumab. In summary, this PhD thesis provides a novel platform for evaluating therapeutic effects on organoids in co-cultures with immune cells. OrganoIDNet, an innovative tool that integrates artificial intelligence with real-time imaging, offers insights into PDAC organoid responses, enhancing personalized drug response assessments. Simultaneously, the co-culture approach using activated T-cells with PDAC PDOs demonstrates its potential to evaluate individual responses of organoids to chemo- and immunotherapeutic on the used case of Mesothelin peptides, emphasizing the importance of personalized strategies in combating PDAC. These insights collectively contribute to developing more effective and tailored therapies for this challenging malignancy.

TPS4232 Background: PDAC is a devastating malignancy. High-throughput genomic technologies have yielded insights regarding the molecular underpinnings and heterogeneity of PDAC. Systemic treatment options are limited to cytotoxic chemotherapies, except for approx. 10%, who receive targeted treatment based on genomic profiling. PDO’s are three-dimensional ex vivo experimental models grown directly from tumor tissue and can provide a direct assessment of drug response. By directly exposing cancer cells to potential drug therapies, functional profiling provides a dynamic measurement of response that is more informative than static gene panels. PDOs can theoretically be used to direct therapeutic decisions, offering an opportunity to expand the reach of precision therapies for PDAC beyond genomics. To date, PDO testing has been limited by small sample sizes, few drugs included in the screens, and retrospective studies. To expand the impact of precision therapy, we developed a rapid high-throughput screening (HTS) platform where over 3,000 drugs can be tested in PDOs within 8-10 weeks of diagnosis. In ADOPT, we aim to formally investigate the efficacy of PDO-directed therapy in a prospective phase II study, leveraging our existing platforms using real-time HTS of PDOs . This study represents one of the first formal trials of PDO-directed therapy in solid tumors. Our novel approach will enroll pts with advanced PDAC who do not have alternative treatment options. Methods: This is an actively recruiting prospective, single-arm phase II trial. Patients (pts) with advanced epithelial PDAC are eligible if they either: 1) progressed on, were intolerant to, or refused first-line or subsequent therapies (Cohort A), or 2) have stable disease after ≥8 cycles of FOLFIRINOX (“Maintenance” Cohort B) and have a PDO showing sensitivity to an approved HC drug. Pts will be recruited, from multiple ongoing studies including PROSPER-PANC where we have successfully generated and tested a PDO. PDO-directed treatment will be selected based on drug sensitivity as tested through our validated HTS platform. Each case will be discussed at our PDO dedicated tumor board. All pts must meet the inclusion/exclusion and drug-specific eligibility criteria. The primary endpoint is disease control rate. A Simon’s two-stage optimal design will be used to test the hypothesis: H0: P ≤ 0.05 versus H1: P ≥ 0.25. In the first stage, 9 pts will be evaluated. The trial will be discontinued if no disease control response is observed in this stage. If at least one response is observed, then the trial will continue to the second stage and an additional 17 pts will be evaluated for a total of 26 evaluable. This design has a one-sided alpha of 0.05 and power of 80%. We will reject the null hypothesis after 26 if 3 or more responses are observed. Clinical trial information: awaited .

Background: Precision oncology entails identification of disease driver mutations and administering drugs that target mutated proteins. However, many breast tumors do not harbor actionable mutations. Even if an actionable mutation is identified, it is unknown if inhibition of the target will lead to clinical response. In metastatic breast cancer, mutation testing alone is insufficiently informative to make optimal treatment selections. FORESEE was a pilot study to assess the feasibility of genomic characterization and functional drug response profiling on patient-derived organoids (PDOs) to prospectively inform therapy selection in metastatic breast cancer (MBC). Methods: Women with newly diagnosed metastatic triple negative (TNBC) or hormone receptor positive/Her2 negative (HR+/Her2-) breast cancer who had exhausted endocrine therapy options were eligible. Patients had to have evaluable disease amenable to fresh biopsy. Tumor tissue was used for single cell sequencing and PDO establishment. PDOs were subjected to screening of drugs approved by the FDA or available to the patient in active clinical trials. Circulating tumor (ct) DNA was also collected. After the biopsy, patients began therapy at the discretion of the treating physician. The results from ct and tumor tissue DNA sequencing were compiled along with results from PDO drug screening in a single report that was discussed with the treating physician. The subsequent line of therapy could align with the recommendation of the report (“informed” therapy) or not (“uninformed therapy”); physicians were not required to follow the recommendations. Response assessments were conducted until documented radiographic or clinical progression. The primary objective was to assess the feasibility of comprehensive genomic characterization and functional drug screening in a clinically relevant timeframe (12 weeks). The efficacy of treatment decisions informed by functional genomic assays constituted an exploratory objective. The trial was registered at clinicaltrials.gov (NCT04450706). Results: 15 patients were enrolled (TNBC, n = 7; HR+/Her2-, n = 8; recurrent, n = 11; de novo metastatic, n = 4). Median age was 55.5 years, median number of prior therapies in the HR+/Her2- patients was 3.5. Tumor biopsies were collected successfully from 13 patients; 10 had adequate tumor content (≥20% tumor content). PDOs were successfully established in 4/10 cases (TNBC, n = 2; HR+/Her2-, n = 2). Drug profiling was successfully conducted in all 4. Functional and genomic results were returned within 12 weeks and informed subsequent therapy selection in all cases. In 3 of 4 cases, genomic testing found no mutations matching FDA approved therapies. In 1 case, actionable mutations in PIK3CA and ESR1 were identified; the treatment choice was arbitrated with PDO drug screening. The relapse-free interval (RFI) with the informed line of therapy did not exceed the RFI with the immediate prior line of therapy. In 2 cases, the informed therapy constituted the last therapy that the patients received. Conclusion: The primary endpoint that PDO-based functional drug screening and genomic characterization will identify clinically actionable outcomes in &amp;gt;46% of patients was not met. Although therapy selection informed by functional drug screening can be clinically meaningful, limitations of this approach included the need for a tumor biopsy with adequate tumor content, low PDO establishment rate, and limited efficacy of approved drugs in advanced MBC. Methodological refinements and generation of PDOs earlier in the natural history of the disease to inform therapy selection may overcome these limitations. Citation Format: C. Vaklavas, L. Zhao, C.-H. Yang, S. D. Scherer, X. Huang, S. S. Buys, M. Wei, P. Moos, G. Marth, B. E. Welm, A. L. Welm. Functional precision oncology for metastatic breast cancer (FORESEE): a feasibility trial. Final Results [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS1-13-20.

e14003 Background: Brain metastases (BrMet) remain a clinically challenge. There is an increase interest in evaluating the efficiacy of systemic therapy for BrMet. Patient-derived organoids (PDO) and xenografts (PDX) are thought to capture the tumor heterogeneity and molecular alterations of the source tumor, and may be used as ‘avatars’ for therapeutic response assessment of the source patient. PDO has an advantage over PDX with a shorter establishment time, and thus, may allow a real-time drug sensitivity testing. The objective of this study was to determine the feasibility of establishing PDO from resected breast cancer BrMet and to evaluate the drug sensitivities in a time period amenable to eventual implemention into clinical utiliity. Methods: Under an IRB-approved protocol, resected BrMet tissues were prospectively collected at the time of clinically indicated neurosurgical procedure as part of a banking program. Tumors were directly cultured as 3-D organoids. DNA and RNA were collected. Drug testing panels included standard clinical care drugs or drug combinations as well as those selected based on the molecular profiling. When clinically conducted next-generation sequencing testing reports were available, we also tested drugs (or class of drugs) that were suggested as a potential therapeutic consideration in these reports. When possible, Nanostring PanCancer Panel evaluation was conducted at the time of PDO establishment to guide the selection of drugs. Results: To date, 11 breast cancer BrMet samples have been collected. 8/11 were successfully established as ‘direct from operating room’ PDO and underwent drug panel testing. The reasons for 3 cases not drug tested include: no viable tumor (confirmed by clinical pathology report as necrosis only) and primary brain tumor (not metastasis). Of the 8 PDO, the clinical subtypes were 5HER2+, 2HR-HER2-, and 1HR+HER2- cases.The median time from collection to the drug testing results availability was 11 days (range: 7-19 days). The median number of drugs or drug combinations tested was 23 (range: 7-32). The drug testing revealed various patterns of sensitivities to chemotherapies and targeted therapies. Nanostring PanCancer testing of PDO identified potential targetable pathways for which PDO demonstrated sensitivity when the drugs were matched to the associated molecular pathway. Conclusions: While the expected number of cases has been much lower due to COVID-19 pandemic-related change in clinical practice patterns and research operations, we have successfully demonstrated that the real-time establishment of BrMet PDO is feasible and may be used as a platform for further investigations. Real-time PDO could be potentially employed to predict drug sensitives for prioritizing drug therapy options in a clinically meaningful time-frame. PDO platform may be used to investigating biomarkers and mechanisms of response and resistance to therapy.