Patient-derived Tumor Samples Research Articles

Abstract B037: Disks large-associated protein 5 (DLGAP5) is a putative prognostic biomarker for intraductal papillary mucinous neoplasm (IPMN) of pancreas

Abstract Pancreatic cancer is one of the deadliest malignant diseases, accounting for nearly 500,000 deaths worldwide each year. One of the pancreatic cancer precursors are mucinous pancreatic cystic lesions (PCL). Although only a minority of these PCLs undergo malignant transformation, the clinical significance is considerable, particularly given the prevalence of up to 45% in individuals over the age of 65. The most prevalent type of neoplastic PCL is intraductal papillary mucinous neoplasm (IPMN) and the management of different IPMN grades varies, with high- grade dysplasia and invasive carcinoma being indicated for surgery. However, the current guidelines for patient stratification are limited and often lead to either overtreatment or undertreatment, both of which are associated with high mortality and morbidity. Therefore, there is a pressing need to identify biomarkers associated with progression of IPMN from low to high grade that can be used for more accurate lesion assessment. Here we identified a novel biomarker highly correlated with IPMN progression – DLGAP5. Both mRNA as well as protein expression of DLGAP5 is positively associated with increased grade of IPMNs as determined by analysis of patient derived tumor samples. Additionally, in vitro experiments confirmed that knock down of DLGAP5 in pancreatic cancer cells leads to reduction of cell proliferation, and conversely, overexpression of DLGAP5 in IPMN precursor cells resulted in increased proliferation. Since one of the functions of DLGAP5 is microtubule stabilization, we hypothesized that the secretome of IPMN precursor cells will be altered by the expression of DLGAP5. Indeed, our mass spectrometry-based analysis of secreted proteins identified several hits (e.g. DKK1) that were correlated with DLGAP5 expression and were previously confirmed to be present in IPMN cyst fluid in patients. Subsequently, we aim to validate the correlation of the putative biomarkers in IPMN cyst fluid and serum with IPMN grade and patients’ clinical outcome in a prospective manner. To sum up, we validated DLGAP5 as an IPMN-progression biomarker and identified several secreted proteins that could serve as DLGAP5-surrogate biomarkers for stratification of the patients with IPMN and subsequent treatment optimalization. Citation Format: Radoslav Janostiak, Peter Mačinga, Lucia Csergeová, Ondřej Fabián, Eva Sticová, Jiří Zahradník, Martin Květoň, Tomáš Hucl. Disks large-associated protein 5 (DLGAP5) is a putative prognostic biomarker for intraductal papillary mucinous neoplasm (IPMN) of pancreas [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B037.

A multiplex single-cell RNA-Seq pharmacotranscriptomics pipeline for drug discovery.

The gene-regulatory dynamics governing drug responses in cancer are yet to be fully understood. Here, we report a pipeline capable of producing high-throughput pharmacotranscriptomic profiling through live-cell barcoding using antibody-oligonucleotide conjugates. This pipeline combines drug screening with 96-plex single-cell RNA sequencing. We show the potential of this approach by exploring the heterogeneous transcriptional landscape of primary high-grade serous ovarian cancer (HGSOC) cells after treatment with 45 drugs, with 13 distinct classes of mechanisms of action. A subset of phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) inhibitors induced the activation of receptor tyrosine kinases, such as the epithelial growth factor receptor (EGFR), and this was mediated by the upregulation of caveolin 1 (CAV1). This drug resistance feedback loop could be mitigated by the synergistic action of agents targeting PI3K-AKT-mTOR and EGFR for HGSOC with CAV1 and EGFR expression. Using this workflow could enable the personalized testing of patient-derived tumor samples at single-cell resolution.

Evaluation of the gene fusion landscape in early onset sporadic rectal cancer reveals association with chromatin architecture and genome stability.

Gene fusions represent a distinct class of structural variants identified frequently in cancer genomes across cancer types. Several gene fusions exhibit gain of oncogenic function and thus have been the focus of development of efficient targeted therapies. However, investigation of fusion landscape in early-onset sporadic rectal cancer, a poorly studied colorectal cancer subtype prevalent in developing countries, has not been performed. Here, we present a comprehensive landscape of gene fusions in EOSRC and CRC using patient derived tumor samples and data from The Cancer Genome Atlas, respectively. Gene Ontology analysis revealed enrichment of unique biological process terms associated with 5'- and 3'- fusion partner genes. Extensive network analysis highlighted genes exhibiting significant promiscuity in fusion formation and their association with chromosome fragile sites. Investigation of fusion formation in the context of global chromatin architecture unraveled a novel mode of gene activation that arose from fusion between genes located in orthogonal chromatin compartments. The study provides novel evidence linking fusions to genome stability and architecture and unearthed a hitherto unidentified mode of gene activation in cancer.

Comparative biological activity of palbociclib and ribociclib in hormone receptor-positive breast cancer

This study examines the biological effects of palbociclib and ribociclib in hormone receptor-positive breast cancer, pivotal to the HARMONIA prospective phase III clinical trial. We explore the downstream impacts of these CDK4/6 inhibitors, focusing on cell lines and patient-derived tumor samples. We treated HR+ breast cancer cell lines (T47D, MCF7, and BT474) with palbociclib or ribociclib (100 nM or 500 nM), alone or combined with fulvestrant (1 nM), over periods of 24, 72, or 144 h. Our assessments included PAM50 gene expression, RB1 phosphorylation, Lamin-B1 protein levels, and senescence-associated β-galactosidase activity. We further analyzed PAM50 gene signatures from the CORALLEEN and NeoPalAna phase II trials. Both CDK4/6 inhibitors similarly inhibited proliferation across the cell lines. At 100 nM, both drugs partially reduced p-RB1, with further decreases at 500 nM over 144 h. Treatment led to reduced Lamin-B1 expression and increased senescence-associated β-galactosidase activity. Both drugs enhanced Luminal A and reduced Luminal B and proliferation signatures at both doses. However, the HER2-enriched signature significantly diminished only at the higher dose of 500 nM. Corresponding changes were observed in tumor samples from the CORALLEEN and NeoPalAna studies. At 2 weeks of treatment, both drugs significantly reduced the HER2-enriched signature, but at surgery, this reduction was consistent only with ribociclib. Our findings suggest that while both CDK4/6 inhibitors effectively modulate key biological pathways in HR+/HER2- breast cancer, nuances in their impact, particularly on the HER2-enriched signature, are dose-dependent, influenced by the addition of fulvestrant and warrant further investigation.

Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.

DIPG-41. CONVECTION ENHANCED DELIVERY-MEDIATED RADIOSENSITIZATION FOR DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children. The only approved standard of care is radiation therapy (RT). Studies examining the role of other systemic agents have failed to demonstrate a survival benefit, partially due to tumor extension to areas of the brain where the blood-brain barrier (BBB) remains intact. As our laboratory has recently determined active NAD(P)H Quinone Dehydrogenase 1 (NQO1) expression in patient-derived DMG tumor samples, we sought to investigate NQO1 bioactivatable agent Napabucasin to augment reactive oxygen species (ROS) production and overcome radioresistance in DMG. Furthermore, we explore the safety and feasibility of convection-enhanced deliver (CED) to overcome limitations of the BBB. METHODS Using two DMG cell lines, we performed clonogenic survival assays with increasing doses of RT with and without Napabucasin. We then created subcutaneous xenograft mouse models, treating with (1) vehicle control, (2) Napabucasin, (3) 2 Gy of RT daily, or (4) combination treatment with RT and Napabucasin. Using a syngeneic orthotopic DMG mouse model, we implanted 7-day osmotic infusion pumps containing either vehicle control or 80uM Napabucasin. Mice then underwent a second randomization with or without daily RT for 5 days. RESULTS We identified a robust radiosensitizing effect with Napabucasin using in vitro clonogenic survival assays. In subcutaneous flank models, combination treatment led to a statistically significant smaller tumor volume post-treatment completion when compared to any monotherapy. In our syngeneic orthotopic DMG mouse model, RT after CED pump implantation in the brainstem did not lead to significant morbidity or mortality. Combination RT and CED of Napabucasin led to a significant survival benefit when compared to monotherapy. CONCLUSIONS In this study, we show Napabucasin as a promising radiosensitizer in DMG. Furthermore, this is the first preclinical study showing safety/efficacy of CED with concurrent RT in DMG.

ETMR-05. DEVELOPMENT OF NOVEL PRECLINICAL MODELS AND THERAPEUTIC STRATEGIES FOR ETMR

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a very rare, aggressive pediatric brain tumor with a dismal 5-year overall survival rate. Few genetic aberrations in miRNA clusters and processing machinery have been detected in ETMRs with 90% of ETMR patients having the C19MC amplification. ETMRs show unique histopathological features which, together with the detection of C19MC or DICER alterations, are necessary for diagnosis. The cellular and molecular mechanisms underlying ETMR development are poorly understood, and targeted therapeutics have not been developed due to scarcity of preclinical models. To expand knowledge of ETMR biology and accelerate the design of novel and therapies, it is thus essential to develop preclinical models. METHODS We have generated two novel patient-derived ETMR cell lines from resected patient derived tumor samples (CTBP-03A and CBTP-139) and created three patient-derived xenograft (PDX) models (CTBP-03A, CBTP-139 and CBTP-38). We have used these unique patient-derived cell lines to identify mechanisms driving ETMR development and progression. To identify novel therapeutic targets, we conducted high-throughput drug screening (HTDS) utilizing 2480 approved and investigational drugs, against the CBTP-03A and CBTP-139 lines. RESULTS Among the candidate drugs showing an inhibitory effect on both lines, compounds were selected based on their activity, CNS penetration ability, mechanism of action, and bioavailability, if known. The top selected hits were screened in an all-versus-all format, totaling 1953 combinations. The rapidity of action and efficacy of the top choice compounds used as single agent or in double and triple combination was assessed by caspase glow and cell titer glow assays. This initial combination screen identified DNA replication stress, retinoid receptor agonist and FGF signaling as potential combinatorial targets. Data are currently being validated in vivo. CONCLUSIONS This is the first combinatorial HTDS on two novel patient-derived cell lines which highlighted potential drug combination strategies for the treatment of ETMR.

Antibodies Targeting Human or Mouse VSIG4 Repolarize Tumor-Associated Macrophages Providing the Potential of Potent and Specific Clinical Anti-Tumor Response Induced across Multiple Cancer Types.

V-set immunoglobulin domain-containing 4 (VSIG4) is a B7 family protein with known roles as a C3 fragment complement receptor involved in pathogen clearance and a negative regulator of T cell activation by an undetermined mechanism. VSIG4 expression is specific for tumor-associated and select tissue-resident macrophages. Increased expression of VSIG4 has been associated with worse survival in multiple cancer indications. Based upon computational analysis of transcript data across thousands of tumor and normal tissue samples, we hypothesized that VSIG4 has an important role in promoting M2-like immune suppressive macrophages and that targeting VSIG4 could relieve VSIG4-mediated macrophage suppression by repolarizing tumor-associated macrophages (TAMs) to an inflammatory phenotype. We have also observed a cancer-specific pattern of VSIG4 isoform distribution, implying a change in the functional regulation in cancer. Through a series of in vitro, in vivo, and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages. Across patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment resulted in the upregulation of cytokines associated with TAM repolarization and T cell activation and chemokines involved in immune cell recruitment. VSIG4 blockade is also efficacious in a syngeneic mouse model as monotherapy as it enhances efficacy in combination with anti-PD-1, and the effect is dependent on the systemic availability of CD8+ T cells. Thus, VSIG4 represents a promising new target capable of triggering an anti-cancer response via multiple key immune mechanisms.

Elucidating the Role of MicroRNA-18a in Propelling a Hybrid Epithelial-Mesenchymal Phenotype and Driving Malignant Progression in ER-Negative Breast Cancer.

Epigenetic alterations that lead to differential expression of microRNAs (miRNAs/miR) are known to regulate tumour cell states, epithelial-mesenchymal transition (EMT) and the progression to metastasis in breast cancer. This study explores the key contribution of miRNA-18a in mediating a hybrid E/M cell state that is pivotal to the malignant transformation and tumour progression in the aggressive ER-negative subtype of breast cancer. The expression status and associated effects of miR-18a were evaluated in patient-derived breast tumour samples in combination with gene expression data from public datasets, and further validated in in vitro and in vivo breast cancer model systems. The clinical relevance of the study findings was corroborated against human breast tumour specimens (n = 446 patients). The down-regulated expression of miR-18a observed in ER-negative tumours was found to drive the enrichment of hybrid epithelial/mesenchymal (E/M) cells with luminal attributes, enhanced traits of migration, stemness, drug-resistance and immunosuppression. Further analysis of the miR-18a targets highlighted possible hypoxia-inducible factor 1-alpha (HIF-1α)-mediated signalling in these tumours. This is a foremost report that validates the dual role of miR-18a in breast cancer that is subtype-specific based on hormone receptor expression. The study also features a novel association of low miR-18a levels and subsequent enrichment of hybrid E/M cells, increased migration and stemness in a subgroup of ER-negative tumours that may be attributed to HIF-1α mediated signalling. The results highlight the possibility of stratifying the ER-negative disease into clinically relevant groups by analysing miRNA signatures.

503 Engineering GBM-specific T Cells Using Photothermal Prussian Blue Nanoparticles

INTRODUCTION: Adoptive T cell therapy (ATCT) has successfully treated hematological malignancies and is currently under investigation for solid tumor therapy. In contrast to existing chimeric antigen receptor (CAR) T cell and/or antigen-specific T cell approaches, which target defined antigens, our PBNP-based approach can target a broad repertoire of antigens in GBM in an antigen-agnostic manner. METHODS: We treated GBM cells with PBNP-based photothermal therapy (PBNP-PTT) prior to culturing with dendritic cells (DCs), and subsequent stimulation of T cells. RESULTS: When PBNP-PTT was administered to U87 cells at an immunogenic thermal dose, we effectively expanded U87-specific T cells. DCs cultured ex vivo with PBNP-PTT-treated U87 cells enabled 15- 30-fold expansion of CD4+ and CD8+ T cells. Upon co-culture with target U87 cells, these T cells secreted IFN-G in a specific and dose-dependent manner (up to 647-fold over controls). T cells manufactured using PBNP-PTT elicited specific cytolytic activity against target U87 cells (33-93% killing at an E:T ratio of 20:1), while sparing normal human astrocytes. Similarly, PBNP-PTT on SNB19 cells resulted in a 7-39-fold expansion of T cells, which elicited 25-66% killing of SNB19 cells at an E:T ratio of 20:1. U87-luc cells were then orthotopically inoculated into NSG mice to establish xenograft GBM, and U87-specific T cells developed via PBNP-PTT were intracranially injected. These T cells eliminated U87 tumors in vivo and significantly improved mouse survival compared to mice treated with PBS (p = 0.0047) or non-specific T cells (p = 0.0123) from the same donor. CONCLUSIONS: These findings provide proof-of-concept data supporting the use of PBNP-PTT to stimulate and expand tumor-specific T cells ex vivo for potential use as an ATCT approach for patients with GBM. We are currently using patient-derived tumor samples to generate IND-enabling data.

Abstract 6423: KRAS mutation detection by Sanger sequencing using the SeqStudio Flex capillary electrophoresis instrument

Abstract The detection of mutations in the KRAS gene highly associated with cancer is one of the most widely used Sanger sequencing capillary electrophoresis (CE) applications in molecular diagnostics. In addition to their traditional role in cancer diagnostic, KRAS mutations have recently gained additional relevance as pharmacogenomics (PGx) markers for a significant number of new oncology therapeutics. KRAS mutation detection assays are used as companion diagnostics (CDx) for these cancer treatments. Analysis by Sanger Sequencing of KRAS mutations is cost effective and lends itself for use in resource limited healthcare settings. While there are currently no commercial kits for targeted sequencing of the KRAS mutation hotspots available, the online Applied Biosystems Primer Designer™ Tool (thermofisher.com) allows standardization of the PCR primer design between labs and repeat orders of the KRAS assays by using these commercially available assay ID numbers. Here we demonstrate the use of Primer Designer KRAS mutation detections assays for mutation in codon 12, 13 and 61 totaling to 8 allele variants. The data was generated on the SeqStudio Flex CE instrument using reference standard DNA as well as DNA extracted from FFPE solid tumor samples. We show accuracy of variant call (OPA 100% (64/64)) and concordance (OPA 98.3% (59/60)) in a side-by-side comparison of the SeqStudio Flex with the legacy Model 3500 Genetic Analyzer instrument using a set of commercially available (Horizon Discovery) KRAS reference DNA samples. Primary solid tumor samples are processed to Fresh Frozen Paraffin Embedded samples (FFPE) for further disease marker evaluation. These samples are stable for long term at ambient temperatures and provide the opportunity to evaluate prospective biomarkers at large scale to understand their potential utility. Discovery Life Sciences has generated a large-scale biorepository of human biospecimen, including FFPE, plasma/sera, buffy coat, and PBMCs. Archival FFPE samples from oncology indications associated with KRAS mutations, such as non-small cell lung cancer and colorectal cancer, were analyzed and identified samples with KRAS mutations, demonstrating the potential diagnostic utility of these assays. Finally, cell-free (cf) DNA from double spun plasma from relevant indications was also successfully evaluated for KRAS mutations, highlighting the potential for these assays in liquid biopsy studies. The study demonstrates the robustness of the Primer Designer KRAS mutation detection sequencing assays using reference standard DNA as well as DNA derived from FFPE DNA and cfDNA from patient derived tumor samples. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Achim Karger, Ami Patel, Kaylee Tseng, Camila Ornelas, Mary Napier, Rod Beck, Kerri Collwell, Shawn Fahl, Carole Bornarth. KRAS mutation detection by Sanger sequencing using the SeqStudio Flex capillary electrophoresis instrument [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 6423.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate >70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [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 2500.

Anaplastic thyroid cancer spheroids as preclinical models to test therapeutics

Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer. Despite advances in tissue culture techniques, a robust model for ATC spheroid culture is yet to be developed. In this study, we created an efficient and cost-effective 3D tumor spheroids culture system from human ATC cells and existing cell lines that better mimic patient tumors and that can enhance our understanding of in vivo treatment response. We found that patient-derived ATC cells and cell lines can readily form spheroids in culture with a unique morphology, size, and cytoskeletal organization. We observed both cohesive (dense and solid structures) and discohesive (irregularly shaped structures) spheroids within the same culture condition across different cell lines. BRAFWT ATC spheroids grew in a cohesive pattern, while BRAFV600E-mutant ATC spheroids had a discohesive organization. In the patient-derived BRAFV600E-mutant ATC spheroids, we observed both growth patterns, but mostly the discohesive type. Histologically, ATC spheroids had a similar morphology to the patient’s tumor through H&E staining and proliferation marker staining. Moreover, RNA sequencing analysis revealed that the gene expression profile of tumor cells derived from the spheroids closely matched parental patient tumor-derived cells in comparison to monolayer cultures. In addition, treatment response to combined BRAF and MEK inhibition in BRAFV600E-mutant ATC spheroids exhibited a similar sensitivity to the patient clinical response. Our study provides a robust and novel ex vivo spheroid model system that can be used in both established ATC cell lines and patient-derived tumor samples to better understand the biology of ATC and to test therapeutics.

Designing patient-oriented combination therapies for acute myeloid leukemia based on efficacy/toxicity integration and bipartite network modeling

Acute myeloid leukemia (AML), a heterogeneous and aggressive blood cancer, does not respond well to single-drug therapy. A combination of drugs is required to effectively treat this disease. Computational models are critical for combination therapy discovery due to the tens of thousands of two-drug combinations, even with approved drugs. While predicting synergistic drugs is the focus of current methods, few consider drug efficacy and potential toxicity, which are crucial for treatment success. To find effective new drug candidates, we constructed a bipartite network using patient-derived tumor samples and drugs. The network is based on drug-response screening and summarizes all treatment response heterogeneity as drug response weights. This bipartite network is then projected onto the drug part, resulting in the drug similarity network. Distinct drug clusters were identified using community detection methods, each targeting different biological processes and pathways as revealed by enrichment and pathway analysis of the drugs’ protein targets. Four drugs with the highest efficacy and lowest toxicity from each cluster were selected and tested for drug sensitivity using cell viability assays on various samples. Results show that ruxolitinib-ulixertinib and sapanisertib-LY3009120 are the most effective combinations with the least toxicity and the best synergistic effect on blast cells. These findings lay the foundation for personalized and successful AML therapies, ultimately leading to the development of drug combinations that can be used alongside standard first-line AML treatment.

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.

T-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells.

The advent of single-cell RNA sequencing (scRNAseq) has enabled in-depth gene expression analysis of several thousand cells isolated from tissues. We recently reported the application of scRNAseq toward the dissection of the tumor-infiltrating T-cell repertoire in human pancreatic cancer samples. In this study, we demonstrated that combined whole transcriptome and T-cell receptor (TCR) sequencing provides an effective way to identify tumor-reactive TCR clonotypes on the basis of gene expression signatures. An important aspect in this respect was the experimental validation of TCR-mediated anti-tumor reactivity by means of an in vitro functional assay, which is the subject of the present protocol. This assay involves the transient transfection of mRNA gene constructs encoding TCRα/β pairs into a well-defined human T-cell line, followed by co-cultivation with the tumor cells of interest and detection of T-cell activation by flow cytometry. Due to the high transfectability and the low background reactivity of the mock-transfected T-cell line to a wide variety of tumor cells, this assay offers a highly robust and versatile platform for the functional screening of large numbers of TCR clonotypes as identified in scRNAseq data sets. Whereas the assay was initially developed to test TCRs of human origin, it was more recently also applied successfully for the screening of TCRs of murine origin. Key features • Efficient functional screening of-and discrimination between-TCRs isolated from tumor-reactive vs. bystander T-cell clones. • Applicable to TCRs from CD8+ and CD4+ tumor-infiltrating T-cells originating from patient-derived tumor samples and syngeneic mouse tumor models. • Rapid flow cytometric detection of T-cell activation by means of TNFα and CD107a expression after a 5 h T-cell/tumor cell co-cultivation.

A Multiplexed Approach to Assess Small Cell Lung Cancer Subtype Heterogeneity in Primary and Patient-Derived Tumor Samples.

Unlocking the heterogeneity of cancers is crucial for developing therapeutic approaches that effectively eradicate disease. As our understanding of markers specific to cancer subclones or subtypes expands, there is a growing demand for advanced technologies that enable the simultaneous investigation of multiple targets within an individual tumor sample. Indeed, multiplex approaches offer distinct benefits, particularly when tumor specimens are small and scarce. Here we describe the utility of two fluorescence-based multiplex approaches; fluorescent Western blots, and multiplex immunohistochemistry (Opal™) staining to interrogate heterogeneity, using small cell lung cancer as an example. Critically, the coupling of Opal™ staining with advanced image quantitation, permits the dissection of cancer cell phenotypes at a single cell level. These approaches can be applied to patient biopsies and/or patient-derived xenograft (PDX) models and serve as powerful methodologies for assessing tumor cell heterogeneity in response to therapy or between metastatic lesions across diverse tissue sites.

Manipulating regulatory T cells: is it the key to unlocking effective immunotherapy for pancreatic ductal adenocarcinoma?

The five-year survival rates for pancreatic ductal adenocarcinoma (PDAC) have scarcely improved over the last half-century. It is inherently resistant to FDA-approved immunotherapies, which have transformed the outlook for patients with other advanced solid tumours. Accumulating evidence relates this resistance to its hallmark immunosuppressive milieu, which instils progressive dysfunction among tumour-infiltrating effector T cells. This milieu is established at the inception of neoplasia by immunosuppressive cellular populations, including regulatory T cells (Tregs), which accumulate in parallel with the progression to malignant PDAC. Thus, the therapeutic manipulation of Tregs has captured significant scientific and commercial attention, bolstered by the discovery that an abundance of tumour-infiltrating Tregs correlates with a poor prognosis in PDAC patients. Herein, we propose a mechanism for the resistance of PDAC to anti-PD-1 and CTLA-4 immunotherapies and re-assess the rationale for pursuing Treg-targeted therapies in light of recent studies that profiled the immune landscape of patient-derived tumour samples. We evaluate strategies that are emerging to limit Treg-mediated immunosuppression for the treatment of PDAC, and signpost early-stage trials that provide preliminary evidence of clinical activity. In this context, we find a compelling argument for investment in the ongoing development of Treg-targeted immunotherapies for PDAC.

Patient-Derived Lung Cancer "Sandwich Cultures" with a Preserved Tumor Microenvironment.

In the past, different spheroid-, organotypic-, and three-dimensional (3D) bioprinting lung cancer models were established for in vitro drug testing and personalized medicine. These tissue models cannot depict the tumor microenvironment (TME) and, therefore, research addressing tumor cell-TME interactions is limited. To overcome this hurdle, we applied patient-derived lung tumor samples to establish new in vitro models. To analyze the tissue model properties, we established two-dimensional (2D) and 3D coculture tissue models exposed to static and dynamic culture conditions that afforded tissue culture for up to 28 days. Our tissue models were characterized by hematoxylin eosin staining, M30 enzyme-linked immunosorbent assay, and immunofluorescence staining against specific lung cancer markers (TTF-1 and p40/p63), cancer-associated fibroblast (CAF) markers (α-SMA and MCT4), and fibronectin (FN). The 3D models were generated with higher success rate than the corresponding 2D model. The cell density of the static 3D model increased from 21 to 28 days, whereas the apoptosis decreased. The dynamic 3D model possessed an even higher cell density than the static 3D model. We identified lung cancer cells, CAFs, and FN. Therefore, a novel in vitro 3D lung cancer model was established, which simulated the TME for 28 days and possessed a structural complexity.

A Novel Patient-Derived Xenograft Model IO-FIVE for Drug Discovery and Precision Medicine in Lymphoma and Leukemia Patients

Lymphoma and leukemia have been the top two hematopoietic cancers in US, leading to more than 40,000 deaths each year. Although the 5-year relative survival rates have been improved up to 60%, there is still a lot of patients showed not response to standard therapy. Patient-Derived Xenograft model (PDX model), as the classical mouse xenograft tumor model that best represents the genetic information characteristics of human tumors, can be used to predict the therapeutic effect, and develop individualized treatment for patients. Its greatest advantage is that the model retains the microenvironment of the original tumor cells, inherits all the molecular biological properties of the primary tumor, and preserves the heterogeneity of the tumor. However, due to the factors that the loss of tumor heterogeneity in hematopoietic cancers after ex vivo clonal expansion and selection, and this in vitro assay based system could not fully reflect the host response to the drugs, including a range of novel immunotherapeutic agents such as PD-1 monoclonal antibody, CD38 monoclonal antibody, etc. With the increasing clinical use of oncology immunological agents, recently we are developing a new assay, named IO-FIVE (Immuno-Oncology drugs Fast In Vivo Efficacy test) for research and therapy, by retaining an appropriate proportion of tumor-infiltrating immune cells (and stromal cells) mixed with tumor cells when digesting clinical tumor samples or patient-derived xenografts-preserved tumor samples into cell suspensions, and then injecting them into the specialized IO-FIVE device, a modified microencapsulation and hollow fiber culture system (OncoVee® MiniPDX), which is implanted subcutaneously into mice and administered systematically for immuno-drug susceptibility testing to screen for immuno-drugs or combinations suitable for individual treatment. The IO-FIVE testing cycle takes only 14 days and is performed using Celltiter Glo before and after the injection of cell suspensions into the specialized device. Celltiter Glo, flow cytometry and Omics (RNA-seq, DNA-seq) are used to measure the viability of total cell subpopulations, the relative ratio of tumor cells to immune cells and the alteration of cellular transcription levels in the device, in order to further explore the sensitivity of tumor tissues to the immunotherapeutic drugs and the potential molecular mechanism for the responders and non-responders. Immune regulatory CD38 antibody Daratumumab has been tested in more than 40 acute myeloid leukemia (AML) cases, with its overall in vivo efficacy around 25%, which is consistent with previous research. It is interesting to uncover novel pathways for drug discovery and clinic treatment since CD38 is well expressed in most AML patients, but at least half of those CD38 + AML patients are not sensitive or resistant to Daratumumab monotherapy. Expression of CD38 on AML is not enough to distinguish the responder vs non-responder of Daratumumab. IO-FIVE could potentially be a companion diagnosis on AML for the patient stratification of Daratumumab. PD1 antibody Sintilimab has been tested in all the patients (12 lymphoma, 52 leukemia), and the only two laboratory PD1-Ab non-responsive T cell lymphoma patients have been showed to be clinically non-responders as well. Immunotherapy and chemical or targeted therapy combination could enhance tumor killing ability and disease control. We will use the IO-FIVE data to further compare it with its source of real-world clinical patients to obtain more evidence-based evidence that can predict individualized treatment with immunotherapy.