Long-term patient-derived ovarian cancer organoids closely recapitulate tumor of origin and clinical response

Organoids, and in particular patient-derived organoids, have emerged as crucial tools for cancer research. Our organoid platform, which has supported patient-derived tumor organoids (PTOs) from a variety of tumor types, has been based on the use of hyaluronic acid (HA) and collagen, or gelatin, hydrogel bioinks. One hurdle to high throughput PTO biofabrication is that as high-throughput multi-well plates, bioprinted volumes have increased risk of contacting the sides of wells. When this happens, surface tension causes bioinks to fall flat, resulting in 2D cultures. To address this problem, we developed an organoid immersion bioprinting method—inspired by the FRESH printing method—in which organoids are bioprinted into support baths in well plates. The bath—in this case an HA solution—shields organoids from the well walls, preventing deformation. Here we describe an improvement to our approach, based on rheological assessment of previous gelatin baths versus newer HA support baths, combined with morphological assessment of immersion bioprinted organoids. HA print baths enabled more consistent organoid volumes and geometries. We optimized the printing parameters of this approach using a cell line. Finally, we deployed our optimized immersion bioprinting approach into a drug screening application, using PTOs derived from glioma biospecimens, and a lung adenocarcinoma brain metastasis. In these studies, we showed a general dose dependent response to an experimental p53 activator compound and temozolomide (TMZ), the drug most commonly given to brain tumor patients. Responses to the p53 activator compound were effective across all PTO sets, while TMZ responses were observed, but less pronounced, potentially explained by genetic and epigenetic states of the originating tumors. The studies presented herein showcase a bioprinting methodology that we hope can be used in increased throughput settings to help automate biofabrication of PTOs for drug development-based screening studies and precision medicine applications.

Only a minority of triple-negative breast cancer (TNBC) patients respond to immune checkpoint inhibitors (ICI). Radiation therapy (RT) increases ICI responses in preclinical TNBC models and in some patients1, 2, but there are no biomarkers to predict RT benefit. TNBC comprises multiple subtypes and we hypothesized that patient-derived tumor organoids (PDOs) representative of such heterogeneity could be used to study RT-induced cancer cell-intrinsic responses.Two PDOs molecularly classified as Luminal Androgen Receptor (LAR) and Basal-Like 1 (BL1) TNBC subtypes3 were mock-treated or irradiated with 3 daily doses of 8Gy and harvested 24 hours post-RT. Differential gene expression analysis (DESeq2), Gene Set Enrichment Analysis (GSEA), and Qiagen’s Ingenuity Pathway Analysis® were performed. Protein expression was assessed by western blot and flow cytometry. Healthy donors CD14+ monocytes were matured in M-CSF for one day prior to culture with PDO. RT upregulated multiple pro-inflammatory pathways (padj < 0.05), including TNFα via NF-κβ, antigen presentation via MHC-I and type I interferon (IFN-I), in the LAR PDO. In contrast, in the BL1 PDO only TNFα via NF-κβ was upregulated by RT, while IFN-I and antigen presentation pathways were not. RT also upregulated genes with immunosuppressive functions (CD73 and TGFB1) in the LAR PDO, and Pathway Analysis identified TGFB1 as a key upstream regulator (p = 7.33 x 10-17). Epithelial to mesenchymal transition (EMT) pathway was induced by RT in both PDOs. Flow cytometry confirmed RT-induced increase in MHC-I and CD73 expression in the LAR PDO, while only CD73 was upregulated in the BL1 PDO. Given the central role of IFN-I induction via cGAS/STING in RT-induced anti-tumor immune responses4, we investigated cGAS and STING expression by the two PDOs. Western blot revealed suppressed expression of STING in the BL1 PDO. STING repression induces resistance to ICI in mouse models of TNBC and was shown to be mediated by MYC amplification in human TNBC5. Consistently, the BL1 PDO was derived from a tumor with MYC amplification. Finally, macrophages co-cultured with the LAR PDO showed reduced MHC-II and CD86 expression, an effect that was reversed when the PDO was treated with RT before co-culture. Our data support the use of PDOs that reflect the heterogeneity of human TNBC as models to study the immunomodulatory effects of RT. Findings show differential induction of key pro-immunogenic mediators by RT in different PDOs and suggest that CD73 is an actionable target to improve RT-induced ICI responses6. Understanding the multifaceted effects of RT using a PDO-based platform may enable a precision use of RT that is tailored to the specific characteristics of a given patient TNBC. Supported by BCRF 23-053 & DOD W81XWH-21-2-0034. DS is the recipient of a Burroughs Welcome Physician-Scientist Award. PMID: 1. 37620372 2. 38194915 3. 27310713 4. 32161398 5. 35561311 6. 32047024 Citation Format: Diego Sialer, Xiaoxue Deng, Maud Charpentier, Neil Rupchand, Marvin Campos, Jenna Moyer, Wen Shen, Olivier Elemento, Doron Betel, Silvia Formenti, Sandra Demaria. Patient-derived triple negative breast cancer organoids show heterogeneous radiation-induced immune signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2194.

The risk of relapse or recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is high despite the combination of surgery and radiochemotherapy, responsible for high toxicity. It is crucial to develop new therapeutic strategies and to identify patients likely to benefit from these treatments. Patient-Derived Tumor Organoids (PDTO) are three-dimensional multicellular structures derived from patient tumor samples and faithfully reproduce the histological and molecular characteristics of the original tumor. A growing body of research indicates that PDTO may predict the clinical response, representing a major opportunity for the development of new therapeutic strategies and precision medicine. The purpose of this translational study is to generate PDTO from HNSCC patients to evaluate their predictive value. PDTO were obtained after dissociation of tumor specimen of HNSCC patients through the setup of the clinical study ORGAVADS (NCT04261192). Tumor cells were embedded in extracellular matrix and cultured in specific medium. Histological and immunohistochemical characterizations were performed to validate the resemblance between PDTO and their original tumor. Response of PDTO to chemotherapy, radiotherapy and innovative therapies were evaluated by live-cell imaging and viability assays. The culture conditions were optimized to improve the success rate of PDTO establishment which now exceeded 50%. Twenty-one PDTO lines have been established and showed histological characteristics close to the original tumor. Expression of immunohistochemical tumor markers p53, p40, p63, and p16 were similar in tumors and paired PDTO. Functional assays were performed on 15 PDTO to analyze the response to treatments (cisplatin, olaparib, X-Rays) and heterogeneity of response was observed between the models. When clinical response was available, PDTO derived from good responders showed high sensitivity to treatments while PDTO from bad responders showed low sensitivity to treatments. Interestingly, two PDTO lines derived from HPV+ oropharyngeal tumors displayed very high sensitivity to cisplatin, matching with the patient's profile and response. Our results showed feasibility to derive PDTO from HNSCC and to perform functional assay to assess their response to different treatments. The first studies of correlation between PDTO and patient responses are promising and will be further investigated in more patients. This will allow to demonstrate the predictive value of PDTO from HNSCC with the purpose to develop a clinically compatible predictive functional assay. Citation Format: Marion Perréard, Vianney Bastit, Lucie Lecouflet, Guillaume Desmartin, Romane Florent, Corinne Jeanne, Juliette Thariat, Emmanuel Babin, Laurent Poulain, Louis-Bastien Weiswald. Establishment of patient-derived tumor organoids from head and neck cancers for predicting response to treatments [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 222.

Automated Scoring to Assess RAD51-Mediated Homologous Recombination in Ovarian Patient-Derived Tumor Organoids.

Fully synthetic nanofibrillar hydrogel for the growth and enzyme-free release of patient-derived ovarian tumor organoids.

Patient-derived tumor organoids (PDOs) are a highly promising preclinical model that recapitulates the histology, gene expression, and drug response of the donor patient tumor. Currently, PDO culture relies on basement-membrane extract (BME), which suffers from batch-to-batch variability, the presence of xenogeneic compounds and residual growth factors, and poor control of mechanical properties. Additionally, for the development of new organoid lines from patient-derived xenografts, contamination of murine host cells poses a problem. We propose a nanofibrillar hydrogel (EKGel) for the initiation and growth of breast cancer PDOs. PDOs grown in EKGel have histopathologic features, gene expression, and drug response that are similar to those of their parental tumors and PDOs in BME. In addition, EKGel offers reduced batch-to-batch variability, a range of mechanical properties, and suppressed contamination from murine cells. These results show that EKGel is an improved alternative to BME matrices for the initiation, growth, and maintenance of breast cancer PDOs.

Triple-negative breast cancer (TNBC, lacking expression of estrogen and progesterone receptors and amplification of HER2) is an aggressive and drug-resistant subtype. Current standard-of-care for early-stage disease with neoadjuvant chemoimmunotherapy results in a pathologic complete response in only ∼60% of patients. Proliferative heterogeneity naturally occurs in most tumors, where tumor cells exist in multiple different states with varying proliferative potential—states of active division, long- and short-term quiescence, pre-cell cycle entry, and stress. TNBC tumors exhibit a higher percentage of actively cycling, or Ki-67–positive cells, compared to other molecular subsets of breast cancer, which has been shown to predict higher pathologic complete response rates following neoadjuvant chemotherapy. Unfortunately, despite this, TNBC patients experience worse overall survival rates driven by higher rates of relapse. It has been proposed that a significant portion of noncycling, or Ki-67–negative, cells are in a G0-like, or quiescent, state and predicted therefore to be resistant to cytotoxic chemotherapies that rely on actively dividing cells. The presence of a tumor cell population in a quiescent/G0-like state has been increasingly recognized across many tumor types, including in breast cancer. Patient-derived tumor organoids have emerged as an appealing model system that retains more cellular heterogeneity from primary tumors compared to widely used two-dimensional cell lines. Organoids show remarkable complexity by single-cell RNA sequencing, representing many more cell states compared to best available two-dimensional cell lines that most closely resemble the mutation, copy number variation, gene expression and protein expression profiles of patient tumors. A mutant version of p27 containing mutations blocking binding to cyclin/cdk complexes, but retained proteolysis in S/G2/M, combined with an mVenus fluorescent protein (p27-mVenus) has been previously validated as a genetic reporter for steady-state G0 readouts. We have optimized lentiviral transduction of a triple-negative breast cancer patient-derived xenograft organoid with the p27-mVenus reporter to label quiescent organoid subpopulations. Optimized parameters include method of lentivirus concentration, multiplicity of infection, duration of transduction, presence of protamine, presence of a Matrigel base layer, presence of RetroNectin and use of spinoculation. We will use this p27-mVenus TNBC organoid reporter line to study the response of quiescent cell populations to current standard of care treatments and how they contribute to therapeutic resistance. Citation Format: Darien Reed-Perino, Sonali Arora, Alana L Welm, Cyrus M Ghajar, Patrick J Paddison. Optimization of lentiviral transduction of a triple-negative breast cancer patient-derived xenograft organoid for modeling tumor cell quiescence and associated treatment resistance [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-04-21.

Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have been approved as monotherapy and combination therapy for BRCA-mutated breast, ovarian, and prostate cancer and it is proposed that they may be effective for other BRCA-mutated cancer as well. However, recent clinical trials show inconsistent efficacy of PARPi on BRCA-mutated cancers, partially due to imperfect biomarker definition and unknown resistance mechanism. BRCA status alone may not be a good indicator for PARPi responsiveness as many proteins are involved in the homologous recombination (HR) pathway, which is why this experiment raises the question of whether time plays a role in tumor drug sensitivity. Patient-derived tumor organoids (PDTOs) can be used to study the pharmacogenomics between PARPis and HR deficient cancer. PDTOs are emerging ex vivo cancer models used for precision medicine because they harbor the mutational landscape of primary tumors. The common pipeline for organoid preclinical research includes confirming mutation profile, performing drug screening, and proposing clinically relevant candidates. The goal of this experiment was to model if the responses of patient-derived colorectal cancer organoids with BRCA gene deletion and TP53, SMAD4, and KRAS gene mutations to the PARPi talazoparib and olaparib differ depending on time.Methods: Patient sample: Colorectal: rectal cancer with BRCA1 gene deletion. Mutations in ⦁ KRAS p.Gly12Val ⦁ TP53 p.Arg175His ⦁ SMAD4 p.Arg361Cys + control: Puromycin 2 ug/mL - control: DMSO PARPi: talazoparib and olaparib In a 96 well plate, 1500 cells per well were plated in 10uL of a media/matrigel mixture and 100 uL of additional media were added to each well. The plate was put in the incubator at 37 C for 48 hours. The PARPi talazoparib and olaparib were then added after a serial dilution at max 50 uM concentration. The plates with the drugs (additional 100uL media with 2X drugs) were put in the incubator at 37 C for 120 hours. The plate for timepoint 1 was then read using the viability assay called Cell Titer Glo 3D. The next timepoint was taken after 168 hours using the same method. In addition, cell viability was measured by high content imaging which is being developed at EIPM.Results: Patient-Derived organoids were sensitive to talazoparib at hour 120 and hour 168 (IC50 1.6 and 1.9 respectively). Patient-derived organoids were not sensitive to olaparib at any time point tested and did not yield a meaningful IC50.Conclusion: The sensitivity of patient-derived colorectal cancer organoids with BRCA gene deletion and TP53, SMAD4, and KRAS gene mutations to the PARPi talazoparib and olaparib did not significantly differ depending on time. However, organoids were more sensitive to talazoparib than to olaparib. These results can guide which PARPi should be used in similar ex vivo experiments to yield meaningful results to eventually lead to successful clinical treatments for patients. Citation Format: Maria Mastropaolo, Helen Kuo, John Nguyen, Florencia Madorsky Rowdo, Jared Capuano, Jenna Moyer, M. Laura Martin, Julianne Hall, Olivier Elemento. Patient-Derived Organoids model time-dependent sensitivities to PARP inhibitors in patients with metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B55.

Purpose:Systemic treatments given to patients with non–small cell lung cancer (NSCLC) are often ineffective due to drug resistance. In the present study, we investigated patient-derived tumor organoids (PDTO) and matched tumor tissues from surgically treated patients with NSCLC to identify drug repurposing targets to overcome resistance toward standard-of-care platinum-based doublet chemotherapy.Experimental Design:PDTOs were established from 10 prospectively enrolled patients with non-metastatic NSCLC from resected tumors. PDTOs were compared with matched tumor tissues by histopathology/immunohistochemistry, whole exome sequencing, and transcriptome sequencing. PDTO growths and drug responses were determined by measuring 3D tumoroid volumes, cell viability, and proliferation/apoptosis. Differential gene expression analysis identified drug-repurposing targets. Validations were performed with internal/external data sets of patients with NSCLC. NSCLC cell lines were used for aldo-keto reductase 1B10 (AKR1B10) knockdown studies and xenograft models to determine the intratumoral bioavailability of epalrestat.Results:PDTOs retained histomorphology and pathological biomarker expression, mutational/transcriptomic signatures, and cellular heterogeneity of the matched tumor tissues. Five (50%) PDTOs were chemoresistant toward carboplatin/paclitaxel. Chemoresistant PDTOs and matched tumor tissues demonstrated overexpression of AKR1B10. Epalrestat, an orally available AKR1B10 inhibitor in clinical use for diabetic polyneuropathy, was repurposed to overcome chemoresistance of PDTOs. In vivo efficacy of epalrestat to overcome drug resistance corresponded to intratumoral epalrestat levels.Conclusions:PDTOs are efficient preclinical models recapitulating the tumor characteristics and are suitable for drug testing. AKR1B10 can be targeted by repurposing epalrestat to overcome chemoresistance in NSCLC. Epalrestat has the potential to advance to clinical trials in patients with drug-resistant NSCLC due to favorable toxicity, pharmacological profile, and bioavailability.

<div>AbstractPurpose:<p>Systemic treatments given to patients with non–small cell lung cancer (NSCLC) are often ineffective due to drug resistance. In the present study, we investigated patient-derived tumor organoids (PDTO) and matched tumor tissues from surgically treated patients with NSCLC to identify drug repurposing targets to overcome resistance toward standard-of-care platinum-based doublet chemotherapy.</p>Experimental Design:<p>PDTOs were established from 10 prospectively enrolled patients with non-metastatic NSCLC from resected tumors. PDTOs were compared with matched tumor tissues by histopathology/immunohistochemistry, whole exome sequencing, and transcriptome sequencing. PDTO growths and drug responses were determined by measuring 3D tumoroid volumes, cell viability, and proliferation/apoptosis. Differential gene expression analysis identified drug-repurposing targets. Validations were performed with internal/external data sets of patients with NSCLC. NSCLC cell lines were used for aldo-keto reductase 1B10 (AKR1B10) knockdown studies and xenograft models to determine the intratumoral bioavailability of epalrestat.</p>Results:<p>PDTOs retained histomorphology and pathological biomarker expression, mutational/transcriptomic signatures, and cellular heterogeneity of the matched tumor tissues. Five (50%) PDTOs were chemoresistant toward carboplatin/paclitaxel. Chemoresistant PDTOs and matched tumor tissues demonstrated overexpression of AKR1B10. Epalrestat, an orally available AKR1B10 inhibitor in clinical use for diabetic polyneuropathy, was repurposed to overcome chemoresistance of PDTOs. <i>In vivo</i> efficacy of epalrestat to overcome drug resistance corresponded to intratumoral epalrestat levels.</p>Conclusions:<p>PDTOs are efficient preclinical models recapitulating the tumor characteristics and are suitable for drug testing. AKR1B10 can be targeted by repurposing epalrestat to overcome chemoresistance in NSCLC. Epalrestat has the potential to advance to clinical trials in patients with drug-resistant NSCLC due to favorable toxicity, pharmacological profile, and bioavailability.</p></div>

<div>AbstractPurpose:<p>Systemic treatments given to patients with non–small cell lung cancer (NSCLC) are often ineffective due to drug resistance. In the present study, we investigated patient-derived tumor organoids (PDTO) and matched tumor tissues from surgically treated patients with NSCLC to identify drug repurposing targets to overcome resistance toward standard-of-care platinum-based doublet chemotherapy.</p>Experimental Design:<p>PDTOs were established from 10 prospectively enrolled patients with non-metastatic NSCLC from resected tumors. PDTOs were compared with matched tumor tissues by histopathology/immunohistochemistry, whole exome sequencing, and transcriptome sequencing. PDTO growths and drug responses were determined by measuring 3D tumoroid volumes, cell viability, and proliferation/apoptosis. Differential gene expression analysis identified drug-repurposing targets. Validations were performed with internal/external data sets of patients with NSCLC. NSCLC cell lines were used for aldo-keto reductase 1B10 (AKR1B10) knockdown studies and xenograft models to determine the intratumoral bioavailability of epalrestat.</p>Results:<p>PDTOs retained histomorphology and pathological biomarker expression, mutational/transcriptomic signatures, and cellular heterogeneity of the matched tumor tissues. Five (50%) PDTOs were chemoresistant toward carboplatin/paclitaxel. Chemoresistant PDTOs and matched tumor tissues demonstrated overexpression of AKR1B10. Epalrestat, an orally available AKR1B10 inhibitor in clinical use for diabetic polyneuropathy, was repurposed to overcome chemoresistance of PDTOs. <i>In vivo</i> efficacy of epalrestat to overcome drug resistance corresponded to intratumoral epalrestat levels.</p>Conclusions:<p>PDTOs are efficient preclinical models recapitulating the tumor characteristics and are suitable for drug testing. AKR1B10 can be targeted by repurposing epalrestat to overcome chemoresistance in NSCLC. Epalrestat has the potential to advance to clinical trials in patients with drug-resistant NSCLC due to favorable toxicity, pharmacological profile, and bioavailability.</p></div>

BACKGROUND:Recent studies have shown patient-derived tumor organoids can predict the drug response of patients with cancer. However, the prognostic value of patient-derived tumor organoid–based drug tests in predicting the progression-free survival of patients with stage IV colorectal cancer after surgery remains unknown.OBJECTIVE:This study aimed to explore the prognostic value of patient-derived tumor organoid–based drug tests in patients with stage IV colorectal cancer after surgery.DESIGN:Retrospective cohort study.SETTINGS:Surgical samples were obtained from patients with stage IV colorectal cancer at the Nanfang Hospital.PATIENTS:A total of 108 patients who underwent surgery with successful patient-derived tumor organoid culture and drug testing were recruited between June 2018 and June 2019.INTERVENTIONS:Patient-derived tumor organoid culture and chemotherapeutic drug testing.MAIN OUTCOMES MEASURES:Progression-free survival.RESULTS:According to the patient-derived tumor organoid-based drug test, 38 patients were drug sensitive and 76 patients were drug resistant. The median progression-free survival was 16.0 months in the drug-sensitive group and 9.0 months in the drug resistant group (p < 0.001). Multivariate analyses showed that drug resistance (HR, 3.38; 95% CI, 1.84–6.21; p < 0.001), right-sided colon (HR, 3.50; 95% CI, 1.71–7.15; p < 0.001), mucinous adenocarcinoma (HR, 2.47; 95% CI, 1.34–4.55; p = 0.004), and non-R0 resection (HR, 2.70; 95% CI, 1.61–4.54; p < 0.001) were independent predictors of progression-free survival. The new patient-derived tumor organoid–based drug test model, which includes the patient-derived tumor organoid–based drug test, primary tumor location, histological type, and R0 resection, was more accurate than the traditional clinicopathological model in predicting progression-free survival (p = 0.001).LIMITATIONS:A single-center cohort study.CONCLUSIONS:Patient-derived tumor organoids can predict progression-free survival in patients with stage IV colorectal cancer after surgery. Patient-derived tumor organoid drug resistance is associated with shorter progression-free survival, and the addition of patient-derived tumor organoid drug tests to existing clinicopathological models improves the ability to predict progression-free survival.

Gallbladder cancer is a highly aggressive malignancy with poor sensitivity to postoperative radiotherapy or chemotherapy; therefore, the development of individualized treatment strategies is paramount to improve patient outcomes. Both patient-derived tumor xenograft (PDX) and patient-derived tumor organoid (PDO) models derived from surgical specimens can better preserve the biological characteristics and heterogeneity of individual original tumors, display a unique advantage for individualized therapy and predicting clinical outcomes. In this study, PDX and PDO models of advanced gallbladder cancer were established, and the consistency of biological characteristics between them and primary patient samples was confirmed using pathological analysis and RNA-sequencing. Additionally, we tested the efficacy of chemotherapeutic drugs, targeted drugs, and immune checkpoint inhibitors using these two models. The results demonstrated that gemcitabine combined with cisplatin induced significant therapeutic effects. Furthermore, treatment with immune checkpoint inhibitors elicited promising responses in both the humanized mice and PDO immune models. Based on these results, gemcitabine combined with cisplatin was used for basic treatment, and immune checkpoint inhibitors were applied as a complementary intervention for gallbladder cancer. The patient responded well to treatment and exhibited a clearance of tumor foci. Our findings indicate that the combined use of PDO and PDX models can guide the clinical treatment course for gallbladder cancer patients to achieve individualized and effective treatment.

BackgroundAlloplex Biotherapeutics has developed a novel autologous cellular therapy for cancer that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand a heterogeneous population of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). The 2-week manufacturing process from PBMCs consistently results 300-fold expansion of NK cells, CD8+ T cells, gamma/delta T cells, NKT cells and some CD4+ T cells, collectively called SUPLEXA therapeutic cells. SUPLEXA cells will be delivered back to cancer patients via intravenous administrations on a weekly schedule as an autologous adoptive cellular immunotherapy for cancer. In this study, we tested SUPLEXA cells developed from normal healthy volunteer PBMCs for their ability to infiltrate and kill patient-derived tumor organoids (PDO) as a pre-clinical assessment for potency against 2 different types of tumor organoids.MethodsTumor organoids derived from colorectal cancer (CRC) or non-small cell lung carcinoma (NSCLC) patients were labeled with cell-trace red dye and plated at equal density in a 96-well plate. After 3 days culture, SUPLEXA cells were thawed (82.8% viable), labeled with cell-trace violet dye, and added to PDO at 1:2 serial diluted numbers ranging from 2 million to 7,800 cells per well. Fluorescent images were captured at 24 hours after adding SUPLEXA cells to PDO models to measure PDO size, tumor infiltration, and PDO killing.ResultsAdding SUPLEXA cells to PDO from CRC and NSCLC resulted in significant infiltration and killing of organoids by 24 hours as shown by the fluorescent images and the organoid size plot for the CRC PDO model (figure 1). Significant reduction in PDO size was observed by adding 31,240 SUPLEXA cells. Similar results were observed with the NSCLC PDO model with significant reduction in PDO size by adding 15,600 SUPLEXA cells. Obvious organoid infiltration was observed in both PDO models and organoid fluorescence was significantly reduced by addition of SUPLEXA cells in both PDO models to suggest that SUPLEXA cells were able to reduce tumor burden (figure 2).Abstract 164 Figure 1CRC organoid infiltration and killing by SUPLEXA. A representative fluorescent image of CRC organoid killing with addition of increasing SUPLEXA cell numbers and a plot showing statistical analysis of 6 replicate wells for changes in CRC organoid size in relation to SUPLEXA cell number additionsAbstract 164 Figure 2Dose-dependent killing in CRC and NSCLC PDO models. CRC and NSCLC organoids were detected by total red fluorescence at 24 hours after adding the indicated numbers of SUPLEXA cells. Loss of red fluorescence after adding SUPLEXA is a measure of overall tumor cell killing/burden in organoids. Data is plotted as mean ± SEM for n=6 replicates per group.ConclusionsSUPLEXA cells infiltrated and killed tumor cells in patient-derived organoids within 24 hours of culture at low cell concentrations indicating potent tumor killing activity. The observed activity in both colorectal and lung cancer organoid models support broad anti-tumor killing activity by SUPLEXA. These results provide further evidence that PBMCs from cancer patients can be activated and expanded by our approach as a novel autologous cellular immunotherapy for cancer.

The tumor microenvironment (TME) of colorectal cancer (CRC) plays an important role in tumor progression and chemoresistance. Therefore, it is important to incorporate elements of the TME, such as extracellular matrix (ECM) composition and stromal cells, into preclinical models to better recapitulate tumor dynamics in vivo. Particularly, cancer-associated fibroblasts (CAFs) are a dominant stromal cell type within the TME that secrete and remodel the ECM and whose abundance can correlate with poor overall survival. Patient-derived tumor organoids (PDTOs) are an innovative preclinical model that has recently emerged as a biomarker for predicting patient-specific drug responses in CRC. However, traditional PDTO cultures fail to account for the aforementioned elements of the TME. Here, we present a basement membrane extract (BME) and collagen type I-based co-culture system utilizing patient-matched CRC PDTOs and CAFs to investigate the effects of the TME on PDTO growth rate, morphology, and drug response.First, we compared the growth rate, morphology, and drug response of PDTO-only cultures in 2 ECMs: 100% BME and 90% BME + 10% collagen type I. PDTOs were seeded 4 days prior to treatment with clinically relevant therapeutics 5-Fluorouracil (5-FU) and SN-38 (active metabolite of irinotecan). Cell viability was assessed 5 days after treatment using the CellTiter-Glo 3D assay. In addition, brightfield and fluorescent images of the PDTOs were taken on days -1, 0, 3, and 5 of the drug treatment. GFP-labeled PDTOs were utilized for the image-based analysis to improve segmentation and quantification of PDTO growth rate and morphology as well as to help distinguish between PDTOs and CAFs in the co-culture experiments. The co-culture experiments of PDTOs and CAFs in 90% BME + 10% collagen type I were performed and analyzed using the same assays. To enhance tumor-stromal interactions, the PDTOs and CAFs were suspended together in the ECM. By culturing CRC PDTOs in the BME + collagen I matrix, as well as co-culturing with patient-matched CAFs, we observed differential responses in PDTO growth rate, morphology, and drug response compared to the 100% BME, PDTO-only condition. In conclusion, our initial findings warrant further investigation into the importance of incorporating elements of the TME into PDTO models for drug discovery applications to more closely mimic in vivo tumor conditions. Citation Format: Scott Valena, Pratiksha Kshetri, Yuyuan Zhao, Elizabeth Elton, Shohei Imamura, Nolan Ung, Seungil Kim, Michael E. Doche, Shannon M. Mumenthaler. The impact of extracellular matrix modulation and cancer-associated fibroblast presence on patient-derived tumor organoid growth rate, morphology, and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 172.