Patient-derived Primary Tumor Research Articles

MDB-19. THE 3D CHROMATIN LANDSCAPE OF MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is a highly malignant childhood cerebellar tumor comprised of four molecularly and clinically distinct subgroups. Sporadic yet subgroup-specific genetic and epigenetic landscapes suggest that higher-order chromatin topology may contribute to distinct subgroup identities and disease biology. Herein, we investigated the 3D chromatin landscape of MB by performing genome-wide chromosome conformation capture (Hi-C) across molecular subgroups using a cohort of n=24 patient-derived xenografts and primary tumor specimens, aiming to elucidate regulatory mechanisms facilitated by genome organization. METHODS Integrative analyses were adapted to incorporate a wide variety of sample-paired datasets. Chromatin contact matrices generated from Hi-C allowed for the mapping of active/inactive compartments (A/B compartments), topologically associated domains (TADs), and distal pairwise chromatin contacts (loops). Topological insights of chromatin architecture were complemented with whole-genome sequencing, RNA-seq, ATAC-seq, DNA methylation, CTCF and histone modification (H3K27ac/H3K27me3) ChIP-seq datasets to dissect mechanisms of gene regulation. RESULTS Unsupervised analysis revealed that chromatin compartmentalization is subgroup stratified. Gene loci contained in subgroup-unique active compartments correspondingly exhibited subgroup-specific chromatin contact patterns, adopted open chromatin conformations, and remained transcriptionally active. As an example, LIN28B resides within a Group 3-specific ‘A’ compartment where its local topology displays higher complexity compared to that of other subgroups. This consequentially facilitates a Group 3-unique loop connecting the LIN28B promoter with a distal enhancer, resulting in subgroup-specific transcriptional upregulation. Targeted displacement of CTCF that anchors and stabilizes chromatin looping at the LIN28B locus was sufficient to alter its transcriptional activity. Using similar lines of investigation, mechanistic insights into the topological determinants of known enhancer-hijacking events, including GFI1, GFI1B and PRDM6, were resolved. CONCLUSIONS Pioneering this integrative approach centered on comprehensive Hi-C maps, our study provides a high-resolution blueprint of the 3D chromatin landscape defining MB subgroups, informing the mechanistic basis of recurrent driver gene activation, and disclosing putative subgroup-specific vulnerabilities.

Methods for assessing and removing non-specific photoimmunotherapy damage in patient-derived tumor cell culture models.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has been shown to selectively destroy tumor in a metastatic mouse model. However, the photoimmunoconjugate (PIC) used for taPIT includes a small fraction of non-covalently associated (free) benzoporphyrin derivative (BPD), which leads to non-specific killing invitro. Here, we report a new treatment protocol for patient-derived primary tumor cell cultures ultrasensitive to BPD photodynamic therapy (BPD-PDT). Based on free BPD efflux dynamics, the updated invitro taPIT protocol precludes non-specific BPD-PDT by silencing the effect of free BPD. Following incubation with PIC, incubating cells with PIC-free medium allows time for expulsion of free BPD whereas BPD covalently bound to PIC fragments is retained. Administration of the light dose after the intracellular free BPD drops below the threshold for inducing cell death helps to mitigate non-specific damage. In this study, we tested two primary ovarian tumor cell lines that are intrinsically chemoresistant, yet ultrasensitive to BPD-PDT such that small amounts of free BPD (afew percent of the total BPD dose) lead to potent induction of cell death upon irradiation. The modifications in the protocol suggested here improve invitro taPIT experiments that lack invivo mechanisms of free BPD clearance (i.e., lymph and blood flow).

Abstract 221: Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies

Abstract Organotypic, three-dimensional (3D) cancer tissue models (OCTM) have enabled investigators to develop predictive markers that can be used in preclinical cancer drug development. However, a drawback of the existing models is that they are mostly based on cell lines based are not physiological. In vitro development of 3D tumoroids using patient derived primary tumors (PDPT) in tumor microenvironment (TME) will have clinical significant and can predict human responses to cancer therapeutics. Here, we describe the generation of OCTM from patient-derived metastatic colorectal cancer, the second most deadly cancer. PDPT were grown in Matrigel for 5 passages and for biobanking purposes tumoroids were further expanded in faCellitate 384 well plates or Sun Bioscience plates to harvest large amounts of well-defined tumoroids cultured for over 21 days. Expanded tumoroids were collected and cryopreserved or gently mixed several times using a pipet tip to break up tumoroids into smaller fragments. These tumor fragments were mixed with endothelial cells, dendritic cells, fibroblasts and embedded in a collagen matrix to simulate TME. The collagen-cell mixture was then added to Transwells and induced to gel to form a collagen gel matrix that simulates TME. The tumoroids were cultured with a specialized medium that supports the growth of complex cell types. The tumor development was monitored daily using bright field microscopy and image analysis was used to monitor tumor size. To evaluate the utility of the OCTM, treatment was initiated at day 10 of the culture period by exposing tissues with the chemotherapeutic drug Cisplatin on days 0, 2, 4, 7, 9, and 11. After each treatment, OCTM tissues (N=2) were fixed for H&E and live/dead staining. Overall, the results of the study showed that: 1) PDPT can be expanded for 5 passages (this low passage number is not expected to change the phenotype of the tumor) and further expanded in high throughput format, 384 well plates, 2) the PDPT could be cultured with multiple cell types in a collagen-gel matrix to recreate the TME, 3) histological evaluation of the OCTM showed a glandular-like tumoroid, 4) immunohistochemical staining revealed that the PDPT in the in vivo-like microenvironment were positive for CK20, and 5) the PDPT shrank following 3 treatment cycles with Cisplatin (day 4). Conclusion: The data shows metastatic colorectal PDPT can be expanded in vitro and cultured in a complex tumor microenvironment on tissue culture inserts. These OCTMs can be used to screen drug safety and efficacy. This study opens new avenues for high-throughput cancer drug screening format using PDPT embedded in a in vivo like TME. This model can be used as a tool for personalized oncology drug testing and for translational assays tailored to other cancer types from our patient derived primary tumor pool. Citation Format: Seyoum Ayehunie, Megan Groves, Bryda Bryda, Alex Armento, Anthony Tolcher. Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies [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 221.

Abstract B036: Micromechanical regulation of tumor immune microenvironment

Abstract Although the prognosis of primary breast cancer (BC) is considerably good, the prognosis of therapy-resistant, metastatic BC remains rather poor, thus being the major cause of BC-related deaths. Due to the undifferentiated and heterogenous nature of this disease, immunotherapies have been among the most promising therapeutic strategies for metastatic BC. However, in recent clinical trials, immunotherapies have had a poor success rate for breast cancer. Therefore, new therapeutic strategies and more profound knowledge of BC-specific immune-evasion mechanisms are urgently needed. We have developed a patient-derived explant model (PDEC), that allows us to cultivate patient-derived primary tumor samples. Moreover, the model also maintains alive the tumor resident immune cells, hence providing us with a unique platform for researching immunological drugs in vivo. In our model, tumor cells and tumor-derived immune cells are embedded in a 3D scaffold matrix with altering biological and physical properties. By altering the material in which the tumor cells are embedded we can also research the effect of microenvironmental factors such as stiffness and growth factors on the tumor cells. Microenvironmental stiffness is a well-known regulator of BC identity; in stiff 3D microenvironment breast cancer cells maintain their original identity, while in a softer microenvironment, they dedifferentiate towards more undifferentiated, therapy-resistant, and metastasis-mimicking subtype (Munne et al, 2021). In this study, we observed for the first time how the alterations in breast tumor microenvironmental stiffness also alter the phenotype in the breast tumor immune microenvironment concomitantly. In the soft microenvironment, the cytokine profile is polarized towards more tumor suppressing via upregulation of TGFB and downregulation of IL1B. Moreover, immune response and antigen presentation are heavily downregulated. Lastly, macrophages are depolarized from M1-like toward more tumor-promoting. This effect is induced and mediated by tumor cells and fresh immune cells embedded in similar 3D conditions that fail to secrete tumor suppressive cytokines or silence antitumor immunity. Manipulation of the tumor cell identity and tumor immune microenvironment via alteration of 3D matrices provides us a unique perspective for observing new methods of how metastatic breast cancer creates immune evasion. Moreover, the model allows us to develop novel treatments against metastatic, theraphy resistant breast cancer and test them easily in ex vivo conditions. Citation Format: Aino Peura, Rita Turpin, Maria Salmela, Ruixian Liu, Piia Mikkonen, Juha Klefström, Pauliina M Munne. Micromechanical regulation of tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B036.

Prediction of Clinical Precision Chemotherapy by Patient-Derived 3D Bioprinting Models of Colorectal Cancer and Its Liver Metastases.

Methods accurately predicting the responses of colorectal cancer (CRC) and colorectal cancer liver metastasis (CRLM) to personalized chemotherapy remain limited due to tumor heterogeneity. This study introduces an innovative patient-derived CRC and CRLM tumor model for preclinical investigation, utilizing 3d-bioprinting (3DP) technology. Efficient construction of homogeneous in vitro 3D models of CRC/CRLM is achieved through the application of patient-derived primary tumor cells and 3D bioprinting with bioink. Genomic and histological analyses affirm that the CRC/CRLM 3DP tumor models effectively retain parental tumor biomarkers and mutation profiles. In vitro tests evaluating chemotherapeutic drug sensitivities reveal substantial tumor heterogeneity in chemotherapy responses within the 3DP CRC/CRLM models. Furthermore, a robust correlation is evident between the drug response in the CRLM 3DP model and the clinical outcomes of neoadjuvant chemotherapy. These findings imply a significant potential for the application of patient-derived 3DP cancer models in precision chemotherapy prediction and preclinical research for CRC/CRLM.

Inserting EF1α-driven CD7-specific CAR at CD7 locus reduces fratricide and enhances tumor rejection.

CAR-T therapies to treat T-cell malignancies face unique hurdles. Normal and malignant T cells usually express the same target for CAR, leading to fratricide. CAR-T cells targeting CD7, which is expressed in various malignant T cells, have limited expansion due to fratricide. Using CRISPR/Cas9 to knockout CD7 can reduce the fratricide. Here we developed a 2-in-1 strategy to insert EF1α-driven CD7-specific CAR at the disrupted CD7 locus and compared it to two other known strategies: one was random integration of CAR by a retrovirus and the other was site-specific integration at T-cell receptor alpha constant (TRAC) locus, both in the context of CD7 disruption. All three types of CD7 CAR-T cells with reduced fratricide could expand well and displayed potent cytotoxicity to both CD7+ tumor cell lines and patient-derived primary tumors. Moreover, EF1α-driven CAR expressed at the CD7 locus enhances tumor rejection in a mouse xenograft model of T-cell acute lymphoblastic leukemia (T-ALL), suggesting great clinical application potential. Additionally, this 2-in-1 strategy was adopted to generate CD7-specific CAR-NK cells as NK also expresses CD7, which would prevent contamination from malignant cells. Thus, our synchronized antigen-knockout CAR-knockin strategy could reduce the fratricide and enhance anti-tumor activity, advancing clinical CAR-T treatment of T-cell malignancies.

Abstract A069: Microscale modeling of the human prostate bone metastatic niche

Abstract Difficulties in modeling the human bone metastasis microenvironment (BMME) niche contribute to limitations in our understanding of the cellular interactions that promote treatment resistance in prostate cancer. To address this, we have developed a multicellular, vascularized, and fully humanized 3D tissue chip model of bone metastases that will allow us to identify drivers of treatment resistance and test emerging cancer therapies in the context of multicellular networks. We utilized LumeNEXT, a closed microfluidics platform with a removable center rod allowing vessel formation, to create this bone microenvironment. To obtain major stromal and bone cell types found in the BMME, we differentiated primary bone marrow-derived mesenchymal stem cells (MSCs) into osteoblasts, fibroblasts, and adipocytes and differentiated patient-derived monocytes from peripheral blood into M2-polarized macrophages and osteoclasts. The primary stromal and bone cells were seeded into LumeNEXT devices and combined in co-cultured with primary prostate cancer tumor organoids in a collagen-based matrix. and deposited into LumeNEXT devices. After polymerization of the hydrogel, the center rod in each device was removed and the tunnels were seeded with endothelial cells to create functional microvasculature. We successfully differentiated the patient-derived cell types, optimized a novel multi-phenotype media (MPM) for co-culture, and maintained greater than 80% viability of all seven cell types in co-culture for at least 14 days in the LumeNEXT platform. qPCR analysis of the model confirmed that expression of differentiation marker genes was maintained after 14 days of co-culture for all cell types. Viability assays and time-lapse microscopy of cell tracker staining also showed viability and growth of prostate cancer organoids in co-culture. Integrated multi-analyte assay platforms were developed for cellular and molecular analysis of patient-derived primary tumor organoids including assessment of cellular phenotype, transcriptomic and epigenetic patterns in the context of multicellular networks. Additionally, we have aimed to develop assays to test emerging cancer-targeting therapies, such as antibody-drug conjugate (ADC). Functional testing of this model using ADC has demonstrated drug perfusion into the stroma through the microvessels, and ADC-specific tumor killing. In summary, we created a multicellular microfluidics model of human bone metastases. High viability of all seven cell types included in our model was maintained for 14 days and early ADC testing demonstrates functional microvasculature and tumor cell killing. The duration of viable co-culture multicellular cultures and promising functional testing demonstrates the suitability of this model for rapid drug testing. Future studies will focus on testing novel chemo-, hormone, and radiation therapies in this multicellular BMME model to determine mechanisms of microenvironment-mediated treatment resistance in prostate cancer. Citation Format: Adeline Ding, Cristina Sanchez de Diego, Sheena C. Kerr, Ravi Chandra Yada, Erika Heninger, Nan Sethakorn, Shannon Reese, Maria Virumbrales-Muñoz, Pete Geiger, Xavier Hazelberg, Joshua M. Lang, Dave Beebe. Microscale modeling of the human prostate bone metastatic niche [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A069.

Abstract 2020: Nascent proteome analysis of drug response in precision-cut tumor slices

Abstract The complex and dynamic microenvironment of tumors influences their development, progression, and response to therapy. In addition to cancer cells, solid tumors consist of a tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. A wide variety of secreted proteins maintain the heterotypic interactions between the cell types in the TME. However, it is not well understood how the TME responds to treatment at the proteome level. Here, we developed a unique nascent proteomic approach for precision-cut tumor slices to address this issue. Precision-cut tumor slices (PCTS) are a technology in which tumor tissues are cut to a defined thickness of 150-300 µm and cultured ex vivo for a certain time. PCTS maintain both the three-dimensional architecture and tumor heterogeneity and preserve their TME with respect to different cell types and the extracellular matrix. Our approach for PCTS nascent proteome analysis combines pulsed-SILAC (stable isotope labeling with amino acids in cell culture) with click chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon drug treatment. PCTS were generated from patient-derived xenografts and primary human ovarian tumors. After a depletion step, the PCTS were cultured in AHA-SILAC medium and treated with cisplatin. PCTS and culture media containing secreted proteins were harvested separately. Newly synthesized proteins were enriched via click chemistry and analyzed using mass spectrometry. A maximum labelling efficiency of >60% was achieved. Human PCTS showed a higher labelling efficiency than mouse xenografts. The PCTS of different tumors showed varying labeling efficiencies, indicating patient heterogeneity. Nascent proteome analysis enables the investigation of drug resistance and response in different patients. Cisplatin treatment resulted in the downregulation of >200 proteins in responsive tumors. A PCTS resistant to cisplatin did not show this response. Moreover, the corresponding patient had a worse clinical outcome than patients whose tumors were sensitive ex vivo. GSEA revealed the involvement of components such as cadherin binding and DNA translation. Protein-protein interactions can be predicted via STRING analysis. Tumor response or resistance was validated using viability assays and immunohistochemical staining for biomarkers of DNA damage and cell death. In conclusion, we established an ex vivo nascent proteome analysis method to study drug response within the complex TME, which can predict the tumor in vivo drug response. By combining the PCTS culture system with pulsed SILAC-AHA treatment, this approach allows the tracking of compositional and dynamic changes within the proteome and monitoring of the direct proteome response on a rapid timescale. It can be used to study cellular communication, predict therapeutic outcomes, and identify new therapeutic targets. Citation Format: Julia Thiel, Lina-Marie Wagner, Karim Aljakouch, Julia Schüler, Bernd Winkler, Kathrin Böpple, Thomas E. Mürdter, Georg Sauer, German Ott, Walter E. Aulitzky, Matthias Schwab, Jeroen Krijgsveld, Meng Dong. Nascent proteome analysis of drug response in precision-cut tumor slices [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 2020.

Rewiring of the 3D genome during acquisition of carboplatin resistance in a triple-negative breast cancer patient-derived xenograft

Changes in the three-dimensional (3D) structure of the genome are an emerging hallmark of cancer. Cancer-associated copy number variants and single nucleotide polymorphisms promote rewiring of chromatin loops, disruption of topologically associating domains (TADs), active/inactive chromatin state switching, leading to oncogene expression and silencing of tumor suppressors. However, little is known about 3D changes during cancer progression to a chemotherapy-resistant state. We integrated chromatin conformation capture (Hi-C), RNA-seq, and whole-genome sequencing obtained from triple-negative breast cancer patient-derived xenograft primary tumors (UCD52) and carboplatin-resistant samples and found increased short-range (< 2 Mb) interactions, chromatin looping, formation of TAD, chromatin state switching into a more active state, and amplification of ATP-binding cassette transporters. Transcriptome changes suggested the role of long-noncoding RNAs in carboplatin resistance. Rewiring of the 3D genome was associated with TP53, TP63, BATF, FOS-JUN family of transcription factors and led to activation of aggressiveness-, metastasis- and other cancer-related pathways. Integrative analysis highlighted increased ribosome biogenesis and oxidative phosphorylation, suggesting the role of mitochondrial energy metabolism. Our results suggest that 3D genome remodeling may be a key mechanism underlying carboplatin resistance.

Replacement, Reduction, and Refinement of Animal Experiments in Anticancer Drug Development: The Contribution of 3D In Vitro Cancer Models in the Drug Efficacy Assessment.

In the last decades three-dimensional (3D) in vitro cancer models have been proposed as a bridge between bidimensional (2D) cell cultures and in vivo animal models, the gold standards in the preclinical assessment of anticancer drug efficacy. 3D in vitro cancer models can be generated through a multitude of techniques, from both immortalized cancer cell lines and primary patient-derived tumor tissue. Among them, spheroids and organoids represent the most versatile and promising models, as they faithfully recapitulate the complexity and heterogeneity of human cancers. Although their recent applications include drug screening programs and personalized medicine, 3D in vitro cancer models have not yet been established as preclinical tools for studying anticancer drug efficacy and supporting preclinical-to-clinical translation, which remains mainly based on animal experimentation. In this review, we describe the state-of-the-art of 3D in vitro cancer models for the efficacy evaluation of anticancer agents, focusing on their potential contribution to replace, reduce and refine animal experimentations, highlighting their strength and weakness, and discussing possible perspectives to overcome current challenges.

Abstract P1-13-23: Carboplatin resistance-associated changes in the 3D chromatin landscape of a triple-negative breast cancer Patient-Derived Xenograft

Abstract Changes in the three-dimensional (3D) structure of the genome are an emerging hallmark of cancer. Cancer-associated copy number variants and single nucleotide polymorphisms promote rewiring of chromatin loops, disruption of topologically associating domains (TADs), active/inactive chromatin state switching, leading to oncogene expression and silencing of tumor suppressors. However, little is known about 3D changes during cancer progression to a chemotherapy-resistant state. We integrated chromatin conformation capture (Hi-C), RNA-seq, and whole-genome sequencing obtained from triple-negative breast cancer patient-derived xenograft primary tumors (UCD52) and carboplatin-resistant samples and found increased short-range (&amp;lt; 2Mb) interactions, chromatin looping, formation of topologically associating domains (TAD), chromatin state switching into a more active state, and amplification of ATP-binding cassette (ABC) transporters. Transcriptome changes suggested the role of long-noncoding RNAs in carboplatin resistance. Rewiring of the 3D genome was associated with TP53, TP63, BATF, FOS-JUN family of transcription factors and led to activation of aggressiveness-, metastasis- and other cancer-related pathways. Integrative analysis highlighted increased ribosome biogenesis and oxidative phosphorylation, suggesting the role of mitochondrial energy metabolism. Our results suggest that 3D genome remodeling may be a key mechanism underlying carboplatin resistance. Citation Format: Mikhail Dozmorov, Maggie Marshall, Narmeen Rashid, Jacqueline Grible, Aaron D. Valentine, Amy Olex, Kavita Murthy, Abhijit Chakraborty, Joaquin Reyna, Daniela Salgado Figueroa, Da-Inn Lee, Brittany Baur, Sushmita Roy, Ferhat Ay, Chuck Harrell. Carboplatin resistance-associated changes in the 3D chromatin landscape of a triple-negative breast cancer Patient-Derived Xenograft [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-23.

Evaluating immune response in vitro in a relevant microenvironment: a high-throughput microfluidic model for clinical screening.

Functional screening of new pharmaceutical compounds requires clinically relevant models to monitor essential cellular and immune responses during cancer progression, with or without treatment. Beyond survival, the emergence of resistant tumor cell clones should also be considered, including specific properties related to plasticity, such as invasiveness, stemness, escape from programmed cell death, and immune response. Numerous pathways are involved in these processes. Defining the relevant ones in the context of a specific tumor type will be key to designing an appropriate combination of inhibitors. However, the diversity and potential redundancy of these pathways remain a challenge for therapy. A new microfluidic device developed by Okomera was dedicated to the screening of drug treatment for breast cancer. This microchip includes 150 droplet-trapping microwells, offering multi-chip settings and multiple treatment choices. After validating the system with established cell lines and a panel of drugs used clinically at Gustave Roussy, preclinical experiments were initiated including patient-derived xenograft (PDX) and primary tumor cells-derived tumoroids with the collaboration of Gustave Roussy clinicians. Tumor-isolated lymphocytes were also added to the tumoroids, using secondary droplets in proof-of-concept experiments. These results show the relevance of the methodology for screening large numbers of drugs, a wide range of doses, and multiple drug combinations. This methodology will be used for two purposes: 1) new drug screening from the compound library, using the high throughput potential of the chip; and 2) pre-clinical assay for a two-weeks response for personalized medicine, allowing evaluation of drug combinations to flag an optimized treatment with potential clinical application.

Comparison of mammosphere formation from stem-like cells of normal breast, malignant primary breast tumors, and MCF-7 cell line

BackgroundMammosphere formation assay has become a versatile tool to quantify the activity of putative breast cancer stem cells in non-adherent in vitro cultures. However, optimizing the suspension culture system is crucial to establish mammosphere cultures from primary breast tumors.MethodsThis study aimed at determining the self-renewal and sphere-forming potential of breast cancer stem-like cells derived from human primary invasive ductal carcinoma and normal breast tissue samples, and MCF-7 breast cancer cell line using an optimal suspension culture system. Mammosphere-forming efficiency of the mammospheres generated from the tissue samples and cell line were compared. We evaluated the expression of CD44+/CD24−/low and CD49f+/EpCAM−/low phenotypes in the stem-like cells by flow cytometry. CK-18, CK-19, α-SMA, and EpCAM marker expression was assessed using immunohistochemical staining.ResultsBreast epithelial cells isolated from the three samples formed two-dimensional spheroids in suspension cultures. Interestingly, mammospheres formed from patient-derived primary breast tumors were enriched in breast cancer stem-like cells with the phenotype CD44+/CD24−/low and exhibited a relatively more number of large spheres when compared to the normal breast stem cells. MCF-7-derived SCs were more aggressive and resulted in the formation of a significantly higher number of spheroids. The expression of CK-18/CK-19 and α-SMA/EpCAM proteins was confirmed in breast cancer tissues.ConclusionsThus, the use of primary tumor specimens and breast cancer cell lines as suitable models for elucidating the breast cancer stem cell activity was validated using mammosphere culture system.

Myeloid checkpoint blockade improves killing of T-acute lymphoblastic leukemia cells by an IgA2 variant of daratumumab.

Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all-trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy.

Nuclear β-catenin translocation plays a key role in osteoblast differentiation of giant cell tumor of bone

Denosumab is a game-changing drug for giant cell tumor of bone (GCTB); however, its clinical biomarker regarding tumor ossification of GCTB has not been elucidated. In this study, we investigated the relationship between Wnt/β-catenin signaling and the ossification of GCTB and evaluated whether endogenous nuclear β-catenin expression predicted denosumab-induced bone formation in GCTB. Genuine patient-derived primary GCTB tumor stromal cells exhibited osteoblastic characteristics. Identified osteoblastic markers and nuclear β-catenin translocation were significantly upregulated via differentiation induction and were inhibited by treating with Wnt signaling inhibitor, GGTI-286, or selective Rac1-LEF inhibitor, NSC23766. Furthermore, we reviewed the endogenous ossification and nuclear β-catenin translocation of 86 GCTB clinical samples and elucidated that intra-tumoral ossification was significantly associated with the nuclear translocation. Three-dimensional quantitative analyses (n = 13) of tumoral CT images have revealed that the nuclear β-catenin translocation of naïve GCTB samples was significantly involved with the denosumab-induced tumor ossification. Our findings suggest a close relationship between the nuclear β-catenin translocation and the osteoblastic differentiation of GCTB. Investigations of the nuclear β-catenin in naïve GCTB samples may provide a promising biomarker for predicting the ossification of GCTB following denosumab treatment.

Abstract 5123: Establishing circulating tumor cell-derived xenografts following a period of in vitro culture

Abstract Cancer metastasis—the spread of cancer cells from a primary node to a distant site—is responsible for as much as 90% of all cancer-associated deaths due to treatment resistance and increased tumor burden. Once established, metastatic nodules oftentimes exhibit distinct molecular phenotypes from their original primary tumor, undermining gold-standard methods for determining treatments for patients, which typically rely on molecular characterization of the primary tumor. Unfortunately, a lack of robust, scalable, and appropriate models for studying differences in patient-derived primary and metastatic tumors has hindered progress in identifying metastasis-specific therapies. Previously, we reported the ability to establish in vitro cultures of circulating tumor cells (CTCs), a unique precursor population of cells that exhibits an ability to escape the primary tumor but not yet fully complete metastasis. We have since expanded the application of this GU-CTC platform to culture CTCs from the blood of patients diagnosed with a variety of metastatic cancers, resulting in the successful culture of 31 unique patient-derived CTC cell lines. Furthermore, following an initial short-term culture period, we also performed subcutaneous injection of mixed CTC cultures into immunocompromised mice, resulting in not only primary tumor formation, but also systemic metastatic nodule formation in as short as 3 months. These resulting CTC-derived xenografts (CDXs) have been demonstrated using metastatic pancreatic, colorectal, and lung adenocarcinoma-derived CTCs. Encouragingly, all CDX models also exhibited replicable metastatic patterns upon reimplantation, reinforcing the important role of CTCs as precursors to metastasis. Bulk RNA-sequencing of patient-matched whole blood, cultured CTCs, and CDX-derived primary tumor and metastatic tissues further identified enrichment of an epithelial-mesenchymal transition as well as several cancer-specific gene signatures consistent with an invasive phenotype. This extended GU-CTC platform therefore enables not only the robust and rapid expansion of CTCs through both in vitro and in vivo methods, but also offers an avenue for personalized drug screens in the effort to target cancer metastasis. Citation Format: Jerry Xiao, Richard Schlegel, Seema Agarwal. Establishing circulating tumor cell-derived xenografts following a period of in vitro culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5123.

Target receptor identification and subsequent treatment of resected brain tumors with encapsulated and engineered allogeneic stem cells

Cellular therapies offer a promising therapeutic strategy for the highly malignant brain tumor, glioblastoma (GBM). However, their clinical translation is limited by the lack of effective target identification and stringent testing in pre-clinical models that replicate standard treatment in GBM patients. In this study, we show the detection of cell surface death receptor (DR) target on CD146-enriched circulating tumor cells (CTC) captured from the blood of mice bearing GBM and patients diagnosed with GBM. Next, we developed allogeneic “off-the-shelf” clinical-grade bifunctional mesenchymal stem cells (MSCBif) expressing DR-targeted ligand and a safety kill switch. We show that biodegradable hydrogel encapsulated MSCBif (EnMSCBif) has a profound therapeutic efficacy in mice bearing patient-derived invasive, primary and recurrent GBM tumors following surgical resection. Activation of the kill switch enhances the efficacy of MSCBif and results in their elimination post-tumor treatment which can be tracked by positron emission tomography (PET) imaging. This study establishes a foundation towards a clinical trial of EnMSCBif in primary and recurrent GBM patients.

Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor.

Chimeric antigen receptor (CAR) T-cell therapy achieves great success for hematological malignancies. However, clinical trials have revealed some limitations in both improving the efficacy and reducing the relapse, which calls for innovative strategies to engineer more powerful CAR-T cells. Promoting the formation of CAR clusters provides an alternative approach and potentially improves current CAR T-cell therapy against cancers. Here, we generated CARCys-T cells using a 4-1BB-derived hinge region including 11 cysteines residues. The cysteines in the hinge were found to facilitate CARCys clustering upon antigen stimulation and promote the antitumor activity of CAR-T cells. Compared with most conventionally used CAR-T cells with CD8α-derived hinge (CARconv-T cells), CARCys-T cells exhibited larger diameter of CAR clusters and enhanced antigen-specific tumor lysis both in vitro and in vivo. In addition, the CARCys-mediated enhancement could be applied to HER2, CD19 as well as GPC3-targeted CAR-T cells. More importantly, CARCys-T cells showed potent antitumor efficacy in clinically relevant patient-derived primary tumor cells and organoids. Thus, the novel hinge containing 11 cysteines provides a promising strategy to facilitate CAR clustering and maximize anti-tumor activity of CAR-T cells, which emphasizes the importance of CAR clustering to improve CAR T-cell therapy in the clinic.

Single-Cell Atlas of Lineage States, Tumor Microenvironment, and Subtype-Specific Expression Programs in Gastric Cancer.

We profiled gastric malignancies at single-cell resolution and identified increased plasma cell proportions as a novel feature of diffuse-type tumors. We also uncovered distinct cancer-associated fibroblast subtypes with INHBA-FAP-high cell populations as predictors of poor clinical prognosis. Our findings highlight potential origins of deregulated cell states in the gastric tumor ecosystem. This article is highlighted in the In This Issue feature, p. 587.

Expansion of the 4-(Diethylamino)benzaldehyde Scaffold to Explore the Impact on Aldehyde Dehydrogenase Activity and Antiproliferative Activity in Prostate Cancer.

Aldehyde dehydrogenases (ALDHs) are overexpressed in various tumor types including prostate cancer and considered a potential target for therapeutic intervention. 4-(Diethylamino)benzaldehyde (DEAB) has been extensively reported as a pan-inhibitor of ALDH isoforms, and here, we report on the synthesis, ALDH isoform selectivity, and cellular potencies in prostate cancer cells of 40 DEAB analogues; three analogues (14, 15, and 16) showed potent inhibitory activity against ALDH1A3, and two analogues (18 and 19) showed potent inhibitory activity against ALDH3A1. Significantly, 16 analogues displayed increased cytotoxicity (IC50 = 10–200 μM) compared with DEAB (>200 μM) against three different prostate cancer cell lines. Analogues 14 and 18 were more potent than DEAB against patient-derived primary prostate tumor epithelial cells, as single agents or in combination treatment with docetaxel. In conclusion, our study supports the use of DEAB as an ALDH inhibitor but also reveals closely related analogues with increased selectivity and potency.