Patient-derived Tumor Explants Research Articles

Different contractility modes control cell escape from multicellular spheroids and tumor explants.

Cells can adapt their active contractile properties to switch between dynamical migratory states and static homeostasis. Collective tissue surface tension, generated among others by the cortical contractility of single cells, can keep cell clusters compact, while a more bipolar, anisotropic contractility is predominantly used by mesenchymal cells to pull themselves into the extracellular matrix (ECM). Here, we investigate how these two contractility modes relate to cancer cell escape into the ECM. We compare multicellular spheroids from a panel of breast cancer cell lines with primary tumor explants from breast and cervical cancer patients by measuring matrix contraction and cellular spreading into ECM mimicking collagen matrices. Our results in spheroids suggest that tumor aggressiveness is associated with elevated contractile traction and reduced active tissue surface tension, allowing cancer cell escape. We show that it is not a binary switch but rather the interplay between these two contractility modes that is essential during this process. We provide further evidence in patient-derived tumor explants that these two contractility modes impact cancer cells' ability to leave cell clusters within a primary tumor. Our results indicate that cellular contractility is an essential factor during the formation of metastases and thus may be suitable as a prognostic criterion for the assessment of tumor aggressiveness.

Abstract B062: CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to therapy. Stromal cancer-associated fibroblasts (CAFs) play a key role in promoting tumor progression and chemoresistance, by creating a fibrotic microenvironment that impedes drug access and feeds PDAC cells nutrients and pro-tumor signals. CRO-67 (Patent PCT/AU2023/050505) is a novel drug developed with Noxopharm Ltd using a rational medicinal chemistry design to improve bioavailability of chromans which have potent anti-cancer activity. Using our patient-derived PDAC tumor in a dish model (explants; maintain multicellular architecture and fibrosis) we previously showed that CRO-67 has both anti-tumor and CAF reprogramming capacity in 4 patient explants [Cancer Res (2022) 82 (22_Supplement):C073]. Aims: 1) To expand evaluation of CRO-67 in additional patient-derived PDAC tumor explants given PDAC heterogeneity. 2) Assess the effect of CRO-67 on PDAC cell and CAF function. 3) Validate the therapeutic potential of CRO-67 on PDAC growth in vivo. Methods: 1) PDAC tumor samples collected from 7 patients undergoing pancreatic resection. Tumor explants (1–2mm diameter) were cultured on gelatin sponges, treated with CRO-67 (0–50μg/mL) every 3 days and fixed on day 12. Therapeutic response assessed by immunohistochemistry for cytokeratin (PDAC cells), α-smooth muscle actin (CAFs), bromodeoxyuridine (proliferation) and TUNEL (cell death). 2) PDAC cells (MiaPaCa-2) and patient-derived CAFs were treated with CRO-67 (1.5μM) for 48h before proliferation analysis (IncuCYTE S3) and for 24h before apoptosis (Annexin V/DAPI staining; flow cytometry) and cell cycle (DAPI staining; flow cytometry) analysis. 3) Subcutaneous PDAC (BxPC3 human cells) tumors in mice were treated with CRO-67 (2.5mg/kg Intraperitoneally, twice a day) for 21 days and tumor volume measured (calipers). Results: 1) CRO-67 treatment decreased tumor and CAF cell frequency in 6/6 (no quantifiable tumor in patient 7 explants) and 7/7 patient PDAC explants, respectively. It also decreased explant cell proliferation and increased cell death versus controls. 2) CRO-67 completely abolished proliferation of MiaPaCa-2 and reduced the average growth rate of CAFs by 81.3±8.8% (p=0.0025; n=5), increased apoptosis in MiaPaCa-2 by 455.7±21.4% (p=0.003; n=3) and in CAFs by 172.6±13.1% (p=0.0067; n=5) and increased the fraction of cells in G2/M cell cycle phase by 163.8±12.8% (p<0.0001; n=3) in MiaPaCa-2 and by 74.6±8.6% (p=0.0015; n=5) in CAFs versus controls. 3) CRO-67 reduced PDAC tumors in vivo by 56.7±6.6% (p=0.0013; n=9 mice/group) versus controls. Conclusions: CRO-67: 1) reduced PDAC cells and CAFs in human PDAC explants via reduced proliferation and increased death; 2) reduced proliferation of PDAC cells and CAFs in vitro, increased apoptosis and disrupted cell cycle progression through G2/M phase; 3) reduced PDAC tumor growth in vivo. Implication: We predict CRO-67 is a potential ‘dual cell’ therapy with direct anti-tumor effects and CAF reprogramming capacity, warranting further investigation in vivo. Citation Format: Keilah Garcia Netto, Shannon Chiang, John Kokkinos, Koroush S. Haghighi, Aparna Raina, Omali Pitiyarachchi, Janet Youkhana, Quach Truong, Daniel Wenholz, Xiang Li, Olivier Laczka, Naresh Kumar, John Wilkinson, David Goldstein, George Sharbeen, Phoebe A. Phillips. CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts [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 B062.

Bioengineered hydrogels enhance ex vivo preservation of patient-derived tumor explants for drug evaluation

Ex vivo patient-derived tumor slices (PDTS) are currently limited by short-term viability in culture. Here, we show how bioengineered hydrogels enable the identification of key matrix parameters that significantly enhance PDTS viability compared to conventional culture systems. As demonstrated using single-cell RNA sequencing and high-dimensional flow cytometry, hydrogel-embedded PDTS tightly preserved cancer, cancer-associated fibroblast, and various immune cell populations and subpopulations in the corresponding original tumor. Cell-cell communication networks within the tumor microenvironment, including immune checkpoint ligand-receptor interactions, were also maintained. Remarkably, our results from a co-clinical trial suggest hydrogel-embedded PDTS may predict sensitivity to immune checkpoint inhibitors (ICIs) in head and neck cancer patients. Further, we show how these longer term-cultured tumor explants uniquely enable the sampling and detection of temporal evolution in molecular readouts when treated with ICIs. By preserving the compositional heterogeneity and complexity of patient tumors, hydrogel-embedded PDTS provide a valuable tool to facilitate experiments targeting the tumor microenvironment.

Changes in Tissue Fluidity Predict Tumor Aggressiveness In Vivo.

Cancer progression is caused by genetic changes and associated with various alterations in cell properties, which also affect a tumor's mechanical state. While an increased stiffness has been well known for long for solid tumors, it has limited prognostic power. It is hypothesized that cancer progression is accompanied by tissue fluidization, where portions of the tissue can change position across different length scales. Supported by tabletop magnetic resonance elastography (MRE) on stroma mimicking collagen gels and microscopic analysis of live cells inside patient derived tumor explants, an overview is provided of how cancer associated mechanisms, including cellular unjamming, proliferation, microenvironment composition, and remodeling can alter a tissue's fluidity and stiffness. In vivo, state-of-the-art multifrequency MRE can distinguish tumors from their surrounding host tissue by their rheological fingerprints. Most importantly, a meta-analysis on the currently available clinical studies is conducted and universal trends are identified. The results and conclusions are condensed into a gedankenexperiment about how a tumor can grow and eventually metastasize into its environment from a physics perspective to deduce corresponding mechanical properties. Based on stiffness, fluidity, spatial heterogeneity, and texture of the tumor front a roadmap for a prognosis of a tumor's aggressiveness and metastatic potential is presented.

The Effects of Natural Epigenetic Therapies in 3D Ovarian Cancer and Patient-Derived Tumor Explants: New Avenues in Regulating the Cancer Secretome.

High mortality rates in ovarian cancer have been linked to recurrence, metastasis, and chemoresistant disease, which are known to involve not only genetic changes but also epigenetic aberrations. In ovarian cancer, adipose-derived stem cells from the omentum (O-ASCs) play a crucial role in supporting the tumor and its tumorigenic microenvironment, further propagating epigenetic abnormalities and dissemination of the disease. Epigallocatechin gallate (EGCG), a DNA methyltransferase inhibitor derived from green tea, and Indole-3-carbinol (I3C), a histone deacetylase inhibitor from cruciferous vegetables, carry promising effects in reprograming aberrant epigenetic modifications in cancer. Therefore, we demonstrate the action of these diet-derived compounds in suppressing the growth of 3D ovarian cancer spheroids or organoids as well as post-treatment cancer recovery through proliferation, migration, invasion, and colony formation assays when compared to the synthetic epigenetic compound Panobinostat with or without standard chemotherapy. Finally, given the regulatory role of the secretome in growth, metastasis, chemoresistance, and relapse of disease, we demonstrate that natural epigenetic compounds can regulate the secretion of protumorigenic growth factors, cytokines, extracellular matrix components, and immunoregulatory markers in human ovarian cancer specimens. While further studies are needed, our results suggest that these treatments could be considered in the future as adjuncts to standard chemotherapy, improving efficiency and patient outcomes.

Abstract A031: A fat lot of good: A novel monounsaturated fatty acid promotes prostate cancer growth and survival

Abstract Advanced prostate cancer (PCa) is currently incurable, and development of novel treatments requires a greater understanding of key pathways that are altered during PCa progression. We have focussed on fatty acid metabolism, as the major source of energy for PCa cells, and have implicated a monounsaturated fatty acid (MUFA), cis-vaccenic acid (cVA), as a critical regulator of PCa cell homeostasis. Production of cVA requires stearoyl-CoA desaturase 1 (SCD1), the enzyme attributed to general MUFA production, and whose activity is important in a range of cancer types. Nonetheless, the specific role of cVA has not previously been studied, in PCa nor cancer more generally. To elucidate the role of cVA in PCa more precisely, we used SCD1 inhibition to study PCa responses to broad MUFA depletion and specific MUFA supplementation, comparing the responses of cVA with its more common structural isomer oleate. To accomplish these aims, we utilised cell line models of PCa (LNCaP, MR49F and V16D) and a benign prostate cell line (PNT1A) to perform cell viability, lipidomic and flow cytometric assays. In addition, responses in clinically-relevant patient-derived tumor explants were measured by proliferative (Ki67) and cell death (CC3) marker staining. Pharmacological SCD1 inhibition (SCDi; A939572) decreased cell viability and increased cell death in PCa cell lines and patient-derived tumor explants, while only minimally affecting the benign prostate cell line PNT1A. Phenotypically, analysis of mitochondrial function and lipidomic changes under SCDi suggested a role for MUFAs in regulating mitochondrial membrane composition via cardiolipins, and oxidative stress levels. This in turn has the potential to regulate cell death, control tumor burden, and influence overall PCa patient survival. Supplementation of cVA or oleate under SCDi demonstrated that only cVA was successful at rescuing cell viability, and cVA, but not oleate, could rescue changes observed in mitochondrial-related lipids, suggesting that cVA regulation of mitochondrial function was a major determinant of its effect on cell viability. Additionally, cVA alone dose-dependently increased PCa cell viability, implicating cVA as an important oncogenic factor. Together, these data identify cVA as a novel substrate required for PCa cell viability, likely via regulation of mitochondrial homeostasis and associated cell death pathways. In summary, this research has revealed more precise insights into the oncogenic effects of an overlooked MUFA, cVA, in PCa, and may uncover new druggable targets that are more selective, to improve treatment options for advanced PCa. Citation Format: Julia S. Scott, Reuben Young, Swati Irani, Jonas Dehairs, Stephen Blanksby, Johannes V. Swinnen, Zeyad D. Nassar, Lisa M. Butler. A fat lot of good: A novel monounsaturated fatty acid promotes prostate cancer growth and survival [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 A031.

Abstract B074: Targeting CBP/p300 and its downstream transcriptional machinery in advanced PCa

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the US. There is a largely unmet clinical need to identify and develop novel strategies, that work either alone or in concert with AR-directed therapeutics, to combat CRPC. The highly conserved histone acetyltransferases CBP/p300 are potent co-activators for AR, and high p300 expression is associated with locally advanced disease and castration-resistant AR function. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC. By employing clinically relevant PCa models, the clinical significance of CBP/p300 expression in PCa patients as well as mechanistic evaluation of CBP/p300 transcriptional reprogramming and DNA damage response pathways have been undertaken. The molecular response to CBP/p300 inhibition will be assessed to discern novel metrics for precision medicine for PCa patients to improve therapeutic efficacy. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. CCS1477 (inobrodib) is a first-in-class bromodomain inhibitor developed by Cell Centric and targeted to inhibit CBP/p300 mediated bromodomain activity, and thus regulate cell survival. Inhibition of the CBP/p300 bromodomain resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression, as well as inhibition of associated factors such c-MYC and its downstream targets, in multiple PCa models. Transcriptomic analysis indicated that both CBP and p300 expression correlate with expression of genes involved in double strand break (DSB) DNA repair process including homologous recombination (HR) and non-homologous end joining (NHEJ) in both primary PCa and CRPC models. CCS1477 directly impacted DNA damage response and repair dynamics, as shown via delay in time to resolution of DNA damage foci formation, including RAD51 and γH2AX foci. Inhibition of CBP/p300 activity decreased tumor cell proliferation, blocked CRPC xenograft growth in vivo, and decreased proliferation ex vivo in patient-derived prostate tumor explants. Importantly, CBP/p300 expression correlated with HR genes in human prostate tissue samples in different cohorts. Lastly, inhibition of CBP/p300 activity also decreased HR gene expression in patients further supporting the essential role CBP/p300 plays in DNA repair. In sum, CBP/p300 inhibition mediates HR repair and impacts patient outcome. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Combined, these studies have the capacity for significant near-term impact in the prevention and/or management of metastatic disease. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati Chand, Wei Yuan, Denisa Bogdan, Jon Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann DeBono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha A. Shafi. Targeting CBP/p300 and its downstream transcriptional machinery in advanced PCa [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 B074.

Dissociation protocols used for sarcoma tissues bias the transcriptome observed in single-cell and single-nucleus RNA sequencing

BackgroundSingle-cell RNA-seq has emerged as an innovative technology used to study complex tissues and characterize cell types, states, and lineages at a single-cell level. Classification of bulk tumors by their individual cellular constituents has also created new opportunities to generate single-cell atlases for many organs, cancers, and developmental models. Despite the tremendous promise of this technology, recent evidence studying epithelial tissues and diverse carcinomas suggests the methods used for tissue processing, cell disaggregation, and preservation can significantly bias gene expression and alter the observed cell types. To determine whether sarcomas – tumors of mesenchymal origin – are subject to the same technical artifacts, we profiled patient-derived tumor explants (PDXs) propagated from three aggressive subtypes: osteosarcoma (OS), Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT). Given the rarity of these sarcoma subtypes, we explored whether single-nuclei RNA-seq from more widely available archival frozen specimens could accurately be identified by gene expression signatures linked to tissue phenotype or pathognomonic fusion proteins.ResultsWe systematically assessed dissociation methods across different sarcoma subtypes. We compared gene expression from single-cell and single-nucleus RNA-sequencing of 125,831 whole-cells and nuclei from ES, DSRCT, and OS PDXs. We detected warm dissociation artifacts in single-cell samples and gene length bias in single-nucleus samples. Classic sarcoma gene signatures were observed regardless of the dissociation method. In addition, we showed that dissociation method biases could be computationally corrected.ConclusionsWe highlighted transcriptional biases, including warm dissociation and gene-length biases, introduced by the dissociation method for various sarcoma subtypes. This work is the first to characterize how the dissociation methods used for sc/snRNA-seq may affect the interpretation of the molecular features in sarcoma PDXs.

Abstract 6647: Dostarlimab shows dose-dependent immune activation of the tumor microenvironment in a patient-derived NSCLC explant model similar to pembrolizumab

Abstract The ability to more faithfully mimic the tumor microenvironment (TME) of cancer patients offers a wide range of possibilities for non-clinical evaluation of anti-cancer therapeutics, including guidance for patient selection, dose, and combinations. We have established a patient-derived tumor explant (PDE) model to monitor immune activation within a structurally preserved TME, following ex vivo incubation with immune check point inhibitors (ICI). When testing anti-PD-1 agents, such as dostarlimab and pembrolizumab, in a cohort of NSCLC explants from stage I-III treatment-naïve patients, we observed that approximately half demonstrate release of inflammatory cytokines after two days incubation. Interestingly, responsive tumors presented with a high level of PD-L1 expression (high TPS), had elevated TMB, and were highly infiltrated by CD8+ T cells at baseline, in line with tumor baseline characteristics predictive of clinical response to anti-PD-1. When testing a dose-range of dostarlimab and pembrolizumab comparable potencies were observed when measuring cytokine release after treatment of the NSCLC explants. In conclusion, we describe here an in vitro patient-derived NSCLC model that mirrors the heterogeneity of patients’ response for anti-PD-1 agents observed in clinical settings, representing a valuable tool for comparative analysis of novel ICI assets and combinations, such as the CD226 axis members. Importantly, this system is also amenable to extensive multi-omics and genetics-based analyses, providing the opportunity to better identify predictive or pharmacodynamic characteristics within patient tumors. Citation Format: Jessica Perrin, Frank Wippich, Valeriia Sherina, Dmytro Dvornikov, Katharina Lupar, Murad M. Melhem, Patrick Hanafin, Jeremy Waight, Jong Yu, Sapna Yadavilli, Daniel Poeckel, Giovanna Bergamini. Dostarlimab shows dose-dependent immune activation of the tumor microenvironment in a patient-derived NSCLC explant model similar to pembrolizumab [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 6647.

Abstract 4501: Novel EHE PDX model used for drug sensitivity

Abstract EHE is a low-to-intermediate grade angiocentric non-vasogenic tumor that exhibits a wide survival range dependent upon tumor size, stage, and location. Most tumors originate in the lung (37%), soft tissue (20%), or liver (16%), and those with multifocal peritoneal or pleural spread have a 5-year survival rate of ~22%. Solitary lesions are often resected for curative intent. However, treatment options for those with metastatic disease at diagnosis remain less well-defined. Generally, the chemo-resistant slow-growing soft-tissue EHEs can often be observed off therapy for years or decades. In contrast, pleural or peritoneal tumors required rapid intervention with systemic cytotoxic chemotherapies. Given the lack of effective treatments, there remains a significant unmet need. Unique among all cancer types, more than 90% of EHEs harbor in-frame chromosomal translocations with WWTR1 (TAZ) or YAP1 in the N-terminal domain. Given that YAP and TAZ are two paralogs, which share numerous epigenetic functions as YAP/TEAD or TAZ/TEAD transcriptional coactivators downstream of the Hippo tumor-suppressor pathway, the oncogenic effect of the resulting EHE-associated TAZ-CAMTA1 (TC) or YAP1-TFE (YT) fusion proteins must, with all certainty, continue to depend on the truncated YAP/TAZ proteins. Since no EHE cell lines exist, one cannot predict if the TC FP behaves similarly in human sarcomas that expose different tissue-specific TEAD binding sites. Trametinib (a MEK inhibitor) was chosen for initial evaluation. We determined the preclinical efficacy of trametinib in the only available EHE patient-derived tumor explant (PDX) in existence, which genetic testing confirmed harbored a TAZ-CAMTA1 translocation. We propagated EHE PDX tumors in mice, then treated with vehicle or trametinib for more than 80 days. Tumor volumes were measured 2-3 times a week, and tumors were collected for further histology and proteomic analysis. Trametinib demonstrated potent antineoplastic activity, consistent with the limited activity observed in recent clinical studies. A separate 10-day study was conducted to measure early pharmacodynamic studies of a potent, oral, selective small molecule palmitoylation inhibitor of TEAD provided by Ikena Oncology. Edge-Seq RNA sequencing and western blots were performed to validate the TEAD-dependent gene and protein expression. EdgeSeq analysis demonstrated significant down-regulation of multiple TEAD dependent genes, indicating on-target inhibition of TEAD in this model. Western blot protein analysis of YAP, TAZ, TEAD1, TC FP, CTGF, and CYR61, collected for pharmacodynamic analysis, demonstrated reduced CYR61, indicative of possible TEAD inhibitor activity; CTGF protein levels were unchanged. The use of one EHE PDX was a limitation, as this model may not fully recapitulate human tumor biology. Future research, using additional EHE PDX models, will be required in gauge the effect of different inhibitors. Citation Format: Sandhya Krishnan, Robert Porter, Danh Truong, Salah-Eddine Lamhamedi-Cherradi, Krzysztof Grela, Rafal Zielinski, Sharon Landers, Vinod Ravi, Joseph A. Ludwig. Novel EHE PDX model used for drug sensitivity [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 4501.

Abstract 1449: Targeting CBP/p300 and its downstream transcriptional machinery in advanced prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the US. There is a largely unmet clinical need to identify and develop novel strategies, that work either alone or in concert with AR-directed therapeutics, to combat CRPC. The highly conserved histone acetyltransferases CBP/p300 are potent co-activators for AR, and high p300 expression is associated with locally advanced disease and castration-resistant AR function. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC. By employing clinically relevant PCa models, the clinical significance of CBP/p300 expression in PCa patients as well as mechanistic evaluation of CBP/p300 transcriptional reprogramming and DNA damage response pathways have been undertaken. The molecular response to CBP/p300 inhibition will be assessed to discern novel metrics for precision medicine for PCa patients to improve therapeutic efficacy. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. CCS1477 (inobrodib) is a first-in-class bromodomain inhibitor developed by Cell Centric and targeted to inhibit CBP/p300 mediated bromodomain activity, and thus regulate cell survival. Inhibition of the CBP/p300 bromodomain resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression, as well as inhibition of associated factors such c-MYC and its downstream targets, in multiple PCa models. Transcriptomic analysis indicated that both CBP and p300 expression correlate with expression of genes involved in double strand break (DSB) DNA repair process including homologous recombination (HR) and non-homologous end joining (NHEJ) in both primary PCa and CRPC models. CCS1477 directly impacted DNA damage response and repair dynamics, as shown via delay in time to resolution of DNA damage foci formation, including RAD51 and γH2AX foci. Inhibition of CBP/p300 activity decreased tumor cell proliferation, blocked CRPC xenograft growth in vivo, and decreased proliferation ex vivo in patient-derived prostate tumor explants. Importantly, CBP/p300 expression correlated with HR genes in human prostate tissue samples in different cohorts. Lastly, inhibition of CBP/p300 activity also decreased HR gene expression in patients further supporting the essential role CBP/p300 plays in DNA repair. In sum, CBP/p300 inhibition mediates HR repair and impacts patient outcome. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Combined, these studies have the capacity for significant near-term impact in the prevention and/or management of metastatic disease. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati Chand, Wei Yuan, Denisa Bogdan, Jon Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann de Bono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha Shafi. Targeting CBP/p300 and its downstream transcriptional machinery in advanced prostate cancer [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 1449.

Abstract 4303: High-throughput 3D tumoroid models for immunotherapy and drug discovery

Abstract The tumor microenvironment (TME) is a complex 3D cellular system comprised of a diverse amalgam of cells. To characterize the TME, we developed a 3D cell culture platform which facilitates imaging-based analysis and cell viability for extended durations. We used this platform to collect in-situ spatiotemporal measurements of cytokine concentrations in the tumor vicinity, track cellular activity, and model local tumor invasion of glioblastoma (GBM) using fast-scanning confocal microscopy. The mechanism by which we achieve long-term 3D culture is perfusion, modeled as flow through a porous medium. Perfusion facilitates regulated transport of nutrients and waste within the soft microgel medium: Liquid-Like Solids (LLS). The interstitial space is tuned to mimic a capillary bed, and surface bioconjugation of the microgels promotes cellular adhesion and migration. To measure cytokine concentrations, GBM tumoroids were grown in LLS and printed into a dispersion of ELISA beads in LLS. The mechanical stability of LLS ensures the tumor and ELISA beads remain stationary without impeding cellular activity. Cytokine on and off-rates were referenced alongside measured bead fluorescence intensities and positions to fit spatiotemporal reaction-diffusion models. Fast-scanning confocal microscopy facilitated in-situ observation of the evolutionary dynamics of tumor progression. Co-culture of patient-derived tumor explants and autologous PBMCs printed into type I collagen-bioconjugated LLS enabled studies of cancer-immune interactions. In-situ cytokine measurements revealed local IL-8 concentrations reached a maximum value of 2 ng ml−1 after 10 hours. A cellular production rate was estimated at 2 molecules cell−1 s−1. Invasive behavior into the proximal space was determined to be super-diffusive; off-lattice agent-based simulations indicated this behavior is a result of the confinement of invasive fronts to the microgel interstitium. The invading glioblastoma cells used anchorage-dependent migration and were guided by geometric cues to traverse the porous bioconjugated LLS network. Cancer-immune interaction studies revealed average CD8+ speeds greater than 2.8 µm min−1 and both chemotaxis and chemokinetic behavior. CD8+ T-cell killing rates were estimated at approximately 3 cancer cells hr−1 initially, monotonically falling over 12 hours to roughly 1 cell hr−1. The development of the in-situ 3D ELISA assay and imaging-based analysis techniques have enabled the tracking of tumor-immune cell interactions, observation of dynamic tumor progression, and local cytokine concentration profiling at appreciable spatiotemporal resolutions. The combination of a physiologically relevant 3D culture platform with the capacity for in-situ qualitative and quantitative observation may lead to new, powerful, preclinical models that allow for interrogation of the TME and decrease the rate of false discovery. Citation Format: Duy Nguyen, Alexander McGhee, Diego Pedro, Alfonso Pepe, Matthew Schaller, Ryan Smolchek, Jack Famiglietti, Stephanie Warrington, W. Gregory Sawyer. High-throughput 3D tumoroid models for immunotherapy and drug discovery. [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 4303.

Abstract 3250: Targeting CD38 on exhausted T cells overcomes resistance to cancer immunotherapy

Abstract Despite the unprecedented success of immune checkpoint blockade (ICB) in melanoma and other cancers, therapeutic resistance remains a major challenge. CD38, an ecto-enzyme involved in NAD+ catabolism, is upregulated in dysfunctional/exhausted CD8+ T cells in human melanoma. As enrichment of dysfunctional CD8+ T cells is associated with lack of response to ICB, and NAD+ depletion via enhanced CD38 activity has been associated with diminished immune response, we hypothesized that CD38 might represent an attractive target to overcome resistance to ICB. While CD38 blockade has been shown to augment response to ICB in murine tumor models, the specific role of CD38 signaling in CD8 T cell exhaustion, and the therapeutic relevance of CD38 in human cancer have not been defined. Here, we confirm and extend previous observations that CD38 is enriched in exhausted CD8+ T cells during tumor progression and in response to ICB. Further, we demonstrate that disrupting CD38 signaling via neutralizing antibodies or pharmacological inhibition enhances response to ICB using established murine tumor models and patient-derived tumor explant models. Notably, we observed 54% response rate to combination PD-1/CD38 blockade using a cohort (n=35) of patient-derived organotypic tumor spheroids (PDOTS) from patients with ICB-refractory melanoma (11.4% response rate to single-agent PD-1 blockade). Supplementation of nicotinamide riboside (NR) to boost NAD+ levels mimicked the effects CD38 blockade/inhibition on PD-1 response, whereas FK866, an inhibitor of NAMPT a key step in NAD+ biosynthesis, blunted the response to combined PD-1/CD38 blockade, suggesting a major role for NAD+ in the efficacy of CD38 blockade/inhibition. Lastly, we demonstrate that in vitro CD38 inhibition/blockade in CD8+ T cells increases the levels of TCF7 (an effector/memory T cell transcription factor), decreases surface expression of co-inhibitory receptors (e.g., TIM-3, CD39, PD-1), and improves effector CD8 T cell function. Taken together, these data confirm a role for CD38 in CD8+ T cell exhaustion in melanoma and support further pre-clinical and clinical development of this novel therapeutic strategy to enhance anti-tumor immune responses. Citation Format: Or-Yam Revach, Angelina M. Cicerchia, Moshe Sade-Feldman, Seth Anderson, Robert T. Manguso, Nir Hacohen, Russell W. Jenkins. Targeting CD38 on exhausted T cells overcomes resistance to cancer immunotherapy [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 3250.

Abstract P2-24-02: Analyzing susceptibility to macrophage-activating immunotherapy bexmarilimab in breast cancer by using patient-derived tumor explants

Abstract The immunosuppressive tumor microenvironment governed by tumor-associated macrophages (TAMs) remains a major obstacle to effective cancer immunotherapy. A novel humanized antibody bexmarilimab, targeting the scavenger receptor Clever-1 on TAMs, has shown clinical benefit in ~40% of patients with late-stage ER+ breast cancer (MATINS; NCT03733990). To increase response rates, a comprehensive understanding on how TAM phenotype and the tumor immune landscape affect bexmarilimab-induced immune activation is needed. Single-cell RNA sequencing analysis of breast tumor macrophages shows high phenotypic variation across pathological subtypes and Clever-1 mRNA (STAB1) expression on various immunosuppressive IL4I1+ and TREM2+ monocyte-derived TAMs. STAB1 expressing macrophages were located in both lymphocyte rich and excluded areas in the tumor stroma based on digital spatial profiling with GeoMx. Due to this diversity, we studied bexmarilimab mode-of-action in breast cancer patient-derived tumor explant cultures (PDEC) by RNA sequencing and cytokine profiling. Our results show that bexmarilimab induced TNFα and CXCL10 secretion in 30-40% of the treated explants. In depth analysis of the responsive PDECs revealed a lower baseline expression of interferon responsive genes in their tumor microenvironment. Correspondingly, in vitro assays with primary human macrophages showed that chronic IFNγ priming abolished bexmarilimab-induced immune activation, suggesting potential use of bexmarilimab in patients with immunologically cold breast tumors. Citation Format: Jenna H. Rannikko, Reetta Virtakoivu, Akira Takeda, Pia Boström, Ilkka Koskivuo, Petri Bono, Maija Hollmén. Analyzing susceptibility to macrophage-activating immunotherapy bexmarilimab in breast cancer by using patient-derived tumor explants [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 P2-24-02.

Making In Vitro Tumor Models Whole Again.

As a reductionist approach, patient-derived in vitro tumor models are inherently still too simplistic for personalized drug testing as they do not capture many characteristics of the tumor microenvironment (TME), such as tumor architecture and stromal heterogeneity. This isespeciallyproblematic for assessing stromal-targeting drugs such as immunotherapies in which the density and distribution of immune and other stromal cells determine drug efficacy. On the other end, in vivo models are typically costly, low-throughput, and time-consuming to establish. Ex vivo patient-derived tumor explant(PDE) cultures involve the culture of resected tumor fragments that potentially retain theintact TME of the original tumor. Although developed decades ago, PDE cultures have not been widely adopted likely because of their low-throughput and poor long-term viability. However, with growing recognition of the importance of patient-specific TME in mediating drug response, especially in the field of immune-oncology, there is an urgent need to resurrect these holistic cultures. In this Review, the key limitations of patient-derived tumor explant cultures are outlined and technologies that have been developed or could be employed to address these limitations are discussed. Engineered holistictumor explant cultures may truly realize the conceptof personalized medicine for cancer patients.

Abstract B076: βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) urgently needs more effective therapies, with a 5-year survival rate <11%. We previously demonstrated βIII-tubulin knockdown in PDAC cells reduced tumor growth and metastasis in vivo, increased chemosensitivity, and induced apoptosis, making it a potent therapeutic target [McCarroll et al, Oncotarget, 6, 2235 (2015)]. However, mechanisms driving these effects are unknown. Apoptosis consists of intrinsic (caspase-9) and extrinsic (caspase-8) pathways. To better understand mechanism and inform therapeutic combinations, our study investigated apoptotic pathways controlled by βIII-tubulin. Aims: 1) Evaluate apoptotic pathways controlled by βIII-tubulin in PDAC cells a) in vitro and b) in vivo; 2) Assess the effect of silencing βIII-tubulin combined with tumor-specific extrinsic apoptosis inducer (TRAIL) in a patient-derived 3D PDAC model. Methods: 1a) PDAC cells were transfected with control-siRNA or βIII-tubulin-siRNA ± caspase-8 or -9 inhibitors and apoptosis measured. The effect of βIII-tubulin knockdown on PDAC cell sensitivity to extrinsic apoptosis inducers (TRAIL, TNFα, FasL) was tested (n≥3). TRAIL induces extrinsic cell death by binding to the TRAIL-receptor DR5, inducing clustering and intracellular activation of caspase 8. DR5 localization following βIII-tubulin knockdown was assessed by confocal microscopy (n=4). Live cell imaging was used to assess trafficking of GFP-tagged DR5 at the membrane of PDAC cells (n≥17 cells/treatment). 1b) Mice with orthotopic PDAC tumors (n=10/group) were treated (5-weeks post-implantation) with nanoparticle+βIII-tubulin-siRNA or control-siRNA for 3.5-weeks. Tumor volume and cleaved caspase-8 were measured. 2) Explants [validated patient-derived 3D tumor model with complete intact stroma, as described in: Kokkinos et al, Sci Rep, 11,1941 (2021); Sharbeen et al, Cancer Res, 81,3461 (2021)] from surgically resected PDAC tumors (5 patients) were cultured for 12 days and treated with nanoparticle+βIII-tubulin-siRNA or control-siRNA every 3 days and TRAIL once weekly. Tumor explants were fixed and assessed for tumor and viability markers via immunohistochemistry. Results: 1a) βIII-tubulin knockdown-induced apoptosis in PDAC cells was blocked by inhibiting caspase-8, but not caspase 9. Knockdown of βIII-tubulin sensitized PDAC cells to extrinsic apoptosis inducers. βIII-tubulin knockdown increased the movement of TRAIL-receptor DR5 through the cell membrane and triggered its clustering in PDAC cells. 1b) βIII-tubulin knockdown in orthotopic PDAC mouse tumors decreased tumor growth and activated extrinsic apoptosis. 2) In patient-derived tumor explants, βIII-tubulin-siRNA plus TRAIL reduced tumor cell frequency. Conclusions: 1) Silencing βIII-tubulin in PDAC cells sensitizes them to extrinsic apoptosis inducers and facilitates increased membrane clustering of the TRAIL receptor DR5. 2) βIII-tubulin knockdown represents an innovative therapeutic strategy to unleash a suicide signal in PDAC cells and render them sensitive to a tumor-specific therapeutic. Citation Format: John Kokkinos, George Sharbeen, Janet Youkhana, Cyrille Boyer, David Goldstein, Koroush S. Haghighi, Joshua A. McCarroll, Phoebe A. Phillips. βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B076.

Abstract C073: CRO-67 is a novel therapeutic for pancreatic cancer: Implications for tumor and stromal reprogramming

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and new therapeutic strategies are urgently required. A key drug delivery barrier and promoter of tumor progression is the fibrotic and heterogeneous microenvironment of PDAC which is mainly orchestrated by stromal cancer-associated fibroblasts (CAFs). Thus, any novel therapeutic strategy should not only target PDAC cells but should also aim to inhibit the pro-tumor properties of the PDAC stroma. However, to accurately evaluate the clinical potential of novel PDAC therapeutics, preclinical models are required that closely mimic the heterogeneity and multicellular architecture of human disease. Aim: We aimed to investigate the therapeutic potential of novel functionalized benzopyrans (CRO-05 & CRO-67) in a patient-derived PDAC tumor explant model. Methods: We collected PDAC tumor samples from patients undergoing resection of their primary tumor. We cultured whole tumor explants (1-2mm diameter) on gelatin sponges. This model accurately maintains the multicellular tumor and stromal architecture of human PDAC [Kokkinos et al, Scientific Reports, 11,1941 (2021)]. Tumor explants were treated every 3 days with the racemic compound CRO-05 or its active enantiomer CRO-67 and explants were fixed in paraformaldehyde at day-12. Therapeutic response was assessed by immunohistochemistry for markers of tumor cells (cytokeratin), CAFs (α-Smooth Muscle Actin), cell-proliferation (Bromodeoxyuridine), and cell-death (TUNEL). Results: Treatment of human PDAC explants with CRO-05 resulted in reduced tumor cell frequency in 3/4 patients and in 4/4 patients, reduced CAF frequency, decreased cell-proliferation, and increased cell-death relative to controls. CRO-67 treatment in explants from 4/5 patients led to reduced tumor cell frequency, and in 5/5 patients, reduced CAF frequency, decreased cell-proliferation, and increased cell-death relative to controls. Conclusions: This work identifies the functionalized benzopyrans CRO-05 and CRO-67 as novel dual-cell therapeutics that potently inhibit both PDAC tumor cells and their surrounding CAFs and provides support for their clinical development as novel PDAC therapeutics. Citation Format: John Kokkinos, George Sharbeen, Koroush S. Haghighi, Janet Youkhana, Aparna Raina, Omali Pitiyarachchi, Quach Truong, Daniel Wenholz, Olivier Laczka, Naresh Kumar, David Goldstein, Phoebe A. Phillips. CRO-67 is a novel therapeutic for pancreatic cancer: Implications for tumor and stromal reprogramming [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C073.

Lupeol and Paclitaxel cooperate in hindering hypoxia induced vasculogenic mimicry via suppression of HIF-1α-EphA2-Laminin-5γ2 network in human oral cancer.

Vasculogenic mimicry (VM), defined as an endothelial cell independent alternative mechanism of blood and nutrient supply by dysregulated tumor cells, is associated with poor prognosis in oral squamous cell carcinoma (OSCC). Here we aim to investigate the underlying molecular mechanism of the synergistic effect of phytochemical Lupeol and standard microtubule inhibitor Paclitaxel in reversing the hypoxia induced VM formation in OSCC. The results demonstrated that the hypoxia induced upregulation of HIF-1α led to augmentation of signaling cascade associated with extracellular matrix remodeling and EMT phenotypes that are mechanistically linked to VM. Induction of HIF-1α altered the expression of EMT/CSC markers (E-Cadherin, Vimentin, Snail, Twist and CD133) and enhanced the ability of cell migration/invasion and spheroid formation. Subsequently, the targeted knockdown of HIF-1α by siRNA led to the perturbation of matrigel mediated tube formation as well as of Laminin-5γ2 expression with the down-regulation of VE-Cadherin, total and phosphorylated (S-897) EphA2, pERK1/2 and MMP2. We also observed that Lupeol in association with Paclitaxel resulted to apoptosis and the disruption of VM associated phenotypes in vitro. We further validated the impact of this novel interventional approach in a patient derived tumor explant culture model of oral malignancy. The ex vivo tumor model mimicked the in vitro anti-VM potential of Lupeol-Paclitaxel combination through down-regulating HIF-1α/EphA2/Laminin-5γ2 cascade. Together, our findings elucidated mechanistic underpinning of hypoxia induced Laminin-5γ2 driven VM formation highlighting that Lupeol-Paclitaxel combination may serve as novel therapeutic intervention in perturbation of VM in human OSCC.

Abstract 180: Automation and high content imaging of 3D triple negative breast cancer patient-derived tumoroids assay for compound screening

Abstract Introduction: Currently there are no clinically approved small molecule targeted therapies for triple negative breast cancer, underlining the critical need to discover new therapeutic targets. Patient-derived cell-based 3D cancer models are highly valuable tools for cancer research and drug development. Primary tumor-derived models can recapitulate tumor heterogeneity and morphology, as well as complex genetic and molecular composition accelerating drug development and drug testing. However, the complexity of performing 3D assays remains a hurdle for adopting these methods for compound screening. In the present study we describe automation of imaging and cell culture methods that enables scaling up complex 3D cell-based assays and compound screening. Methods: We developed an integrated workcell that includes a confocal imaging system, an automated CO2 incubator, an automated liquid handler, and a Collaborative Robot combining different instruments. Workcell allows automation of compound testing, culture monitoring, and evaluation of phenotypic effects of drugs with high content imaging. Tumoroids were formed from primary cells isolated from a patient-derived tumor explant, TU-BcX-4IC, that represents metaplastic breast cancer with a triple-negative breast cancer subtype. Cells were expanded in 2D format, then tumoroids were formed by plating 2,000 cells per well in U-shape low attachment 384 plates. Tumoroids (200 μm in diameter) were formed 48 hr after plating and were treated with 165 compounds of approved cancer drugs from the NCI library at multiple concentrations (10 nM to 100 μM). Compound dilutions, additions, media additions, and staining were performed using liquid handler. During incubation with compounds, tumoroids were monitored daily using transmitted light imaging. For end point assay tumoroids were stained with viability dyes and imaged using automated confocal imaging system. Advanced image analysis included 3D reconstitution and complex phenotypic evaluation of tumoroids. Results: We characterized multiple quantitative descriptors that can be used for studying tumor phenotypes and compound effects including characterization of size and integrity, cell morphology and viability, as well as determining presence and expression levels for various cell markers. Concentration-dependent effects of numerous compounds were observed across multiple read-outs that indicate cytotoxic effects, with 8 compounds demonstrating effects at 10 nM (e.g, romidepsin, trametinib, bortezomib, carfilzomib, and panobinostat). These compounds include HDAC inhibitors and proteosome inhibitors. Conclusions: We demonstrated methods for increased throughput and automation in 3D cancer assays and compound screening, and also analysis approaches and descriptors that provide information about cell phenotypes and compound effects. Citation Format: Oksana Sirenko, Angeline Lim, Courtney K. Brock, Katya Nikolov, Cathy Olsen, Evan F. Cromwell, Margarite D. Matossian, Matthew E. Burow. Automation and high content imaging of 3D triple negative breast cancer patient-derived tumoroids assay for compound screening [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 180.

Multifunctional profiling of triple-negative breast cancer patient-derived tumoroids for disease modeling.

3D cell models derived from patient tumors are highly translational tools that can recapitulate the complex genetic and molecular compositions of solid cancers and accelerate identification of drug targets and drug testing. However, the complexity of performing assays with such models remains a hurdle for their wider adoption. In the present study, we describe methods for processing and multi-functional profiling of tumoroid samples to test compound effects using a novel flowchip system in combination with high content imaging and metabolite analysis. Tumoroids were formed from primary cells isolated from a patient-derived tumor explant, TU-BcX-4IC, that represents metaplastic breast cancer with a triple-negative breast cancer subtype. Assays were performed in a microfluidics-based device (Pu⋅MA System) that allows automated exchange of media and treatments of tumoroids in a tissue culture incubator environment. Multi-functional assay profiling was performed on tumoroids treated with anti-cancer drugs. High-content imaging was used to evaluate drug effects on cell viability and expression of E-cadherin and CD44. Lactate secretion was used to measure tumoroid metabolism as a function of time and drug concentration. Observed responses included loss of cell viability, decrease in E-cadherin expression, and increase of lactate production. Importantly, the tumoroids were sensitive to romidepsin and trametinib, while showed significantly reduced sensitivity to paclitaxel and cytarabine, consistent with the primary tumor response. These methods for multi-parametric profiling of drug effects in patient-derived tumoroids provide an in depth understanding of drug sensitivity of individual tumor types, with important implications for the future development of personalized medicine.