Human Tumor Tissues Research Articles

Injectable and Sprayable Fluorescent Nanoprobe for Rapid Real-Time Detection of Human Colorectal Tumors.

The development of minimally invasive surgery has greatly advanced precision tumor surgery, but sometime suffers from restricted visualization of the surgical field, especially during the removal of abdominal tumors. A 3-D inspection of tumors could be achieved by intravenously injecting tumor-selective fluorescent probes, whereas most of which are unable to instantly distinguish tumors via in situ spraying, which is urgently needed in the process of surgery in a convenient manner. In this study, this work has designed an injectable and sprayable fluorescent nanoprobe, termed Poly-g-BAT, to realize rapid tumor imaging in freshly dissected human colorectal tumors and animal models. Mechanistically, the incorporation of γ-glutamyl group facilitates the rapid internalization of Poly-g-BAT, and these internalized nanoprobes can be subsequently activated by intracellular NAD(P)H: quinone oxidoreductase-1 to release near-infrared fluorophores. As a result, Poly-g-BAT can achieve a superior tumor-to-normal ratio (TNR) up to 12.3 and enable a fast visualization (3min after in situ spraying) of tumor boundaries in the xenograft tumor models, Apcmin/+ mice models and fresh human tumor tissues. In addition, Poly-g-BAT is capable of identifying minimal premalignant lesions via intravenous injection.

CNSC-04. EXPLORATION OF INTERCELLULAR NETWORKS IN EPENDYMOMA REVEALS POTENTIAL THERAPEUTIC VULNERABILITIES

Abstract BACKGROUND Aggressive ependymoma (EPN) exhibit primary and secondary radio- and chemotherapy resistance with the poorest prognosis in the pediatric groups PF-EPN-A and ST-EPN-ZFTA. Recently, tumor cell networks formed by membrane protrusions, such as tunneling nanotubes (TNT) or tumor microtubes (TM), have been identified in other glial brain tumors. These structures facilitate intercellular hierarchical Ca2+-communication and contribute to therapy resistance. Previous data from our group suggested enhancer-regulated Ca2+-communication to be essential in EPN. This study aims to further characterize intratumoral interactions and to understand potential correlations between tumor cell connections and treatment resistance in EPN. METHODS Intercellular connections in EPN patients and model systems were characterized by scanning electron microscopy, immunofluorescence and ultra-high content imaging. Transcriptomic and proteomic data were analyzed to identify potential targetable vulnerabilities within network dynamics. Candidates were functionally validated in cell culture models applying Ca2+ live cell imaging. RESULTS Connectivity-related terms such as microtube-based movement or microtube bundle formation showed increased expression in EPN transcriptome data compared to healthy controls. EPN networks were found to be build of TM- and TNT-like structures with a higher abundance of TNT-like structures in PF-EPN-A compared to ST-EPN-ZFTA. All networks showed strong Nestin-positivity in cells, mouse models and human tumor tissue of ST-EPN-ZFTA and PF-EPN-A. Thrombospondin-1, a matricellular protein enhancing TMs in glioma cells, was strongly enriched in ST-EPN-ZFTA patients. The gap junction channel connexin 43, connecting glioma cells, was highly upregulated in PF-EPN-A. Active electrochemical communication was observed between ST-EPN-ZFTA cells performing Ca2+ imaging. Moreover, inhibition of T-type Ca2+ channels impaired intercellular signaling, leading to network perturbation in vitro. CONCLUSIONS Our study revealed the importance of gap junction-coupled networks for communication of aggressive EPN cells. Targeted disconnection of the EPN-specific network as demonstrated for Ca2+ channel inhibition suggests a new avenue for therapeutic strategies in EPN to overcome resistance.

LGG-33. NETWORK ANALYSIS INTEGRATING MICRORNAS AND CLINICAL DATA TO DETERMINE PROGNOSTIC OUTCOME IN PEDIATRIC LOW-GRADE GLIOMA PATIENTS

Abstract BACKGROUND Low-grade pediatric gliomas (pLGGs) are the most common pediatric brain tumors. Surgical resection, when feasible, represents the standard of care. If complete resection cannot be achieved, disease progression occurs in about 30% of the cases, and these patients receive adjuvant therapy. The identification of predictive biomarkers to predict tumors that will progress after partial resection, requiring systemic treatment (Chemotherapy or target therapy), could support clinicians in the decision-making strategy. MicroRNAs have been related to tumorigenesis and tumor maintenance and can be used as important biomarkers. This study aims to develop a predictive model able to determine prognostic outcomes and to better stratify pLGG patients according to disease progression. METHODS MicroRNAs levels have been assessed in 76 human tumor tissue samples. Clinical data have been collected for statistical and network-based analyses. Using a network-based approach, we integrated clinical data and tissue microRNA expression to identify a microRNA signature able to predict disease progression. RESULTS Our preliminary microRNA expression analysis displayed new promising signatures correlating with prognostic features, such as disease progression and embryological origin of the tumor. CONCLUSION The predictive model we developed integrating biomarkers and clinical data could be crucial to support clinicians in the decision-making strategy and in advancing personalized medicine.

Comparison of primary and passaged tumor cell cultures and their application in personalized medicine.

Passaged cell lines represent currently an integral component in various studies of malignant neoplasms. These cell lines are utilized for drug screening both in monolayer cultures or as part of three-dimensional (3D) tumor models. They can also be used to model the tumor microenvironment in vitro and in vivo through xenotransplantation into immunocompromised animals. However, immortalized cell lines have some limitations of their own. The homogeneity of cell line populations and the extensive passaging in monolayer systems make these models distant from the original disease. Recently, there has been a growing interest among scientists in the use of primary cell lines, as these are passaged directly from human tumor tissues. In this case, cells retain the morphological and functional characteristics of the tissue from which they were derived, an advantage often not observed in passaged cultures. This review highlights the advantages and limitations of passaged and primary cell cultures, their similarities and differences, as well as existing test systems that are based on primary and passaged cell cultures for drug screening purposes.

Abstract PO4-27-12: Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence

Abstract Background: Resistance to endocrine therapy (ET; tamoxifen, aromatase inhibitors, AI, or fulvestrant) in ER+ breast cancer (BC) could be due to survival of breast cancer stem cells (BCSCs). BCSCs contribute to relapse of ER+ BC. We discovered a novel and potent anti-BCSC gene, Death Associated Protein 6 (DAXX) through a pre-surgical biomarker window study combining ET plus a Notch inhibitor [MK-0752, a g-secretase inhibitor (GSI)]. We also found that ET enhances the BCSC population by decreasing DAXX protein levels. In this current study, we measured DAXX protein levels in ER+ PDX tumors and human tumor tissues treated with ET. We determined the mechanism by which ET regulates DAXX expression at the level of protein stability. The goal of this study was to identify novel therapeutic strategies to prevent ET-mediated degradation of the DAXX protein, eliminate BCSCs, and prevent ER+ recurrence. Methods: ER+ BC cell lines (MCF-7 and T47D), an ER+ PDX BCM 5097 tumor line, and human breast cancer tumor samples were used. The ER+ PDX tumor (BCM 5097) was implanted into NSG mice and passaged into female athymic, nude mice-supplemented with an estradiol capsule. After one week, the estradiol capsule was removed from 10 mice, to mimic the use of AI. The remaining 10 mice retained the estradiol capsule. Tumor area was measured weekly up to 50 weeks. Western blotting detected ERa, the stem cell factor Notch4, and DAXX levels. Immunohistochemistry (IHC) detected DAXX protein levels. ER+ human tumor samples were collected prior to and after neoadjuvant treatment with ET (N=11) or chemotherapy (N=14) and stained for the DAXX protein. Correlations were made between DAXX and Ki67. Full length and deletion mutants of DAXX were expressed in ER+ BC cells. BCSCs were measured using the mammosphere forming assay. Liquid chromatography followed by mass spectrometry were used to detect phosphorylated residues of DAXX in response to ET. Kinases that phosphorylate these residues on DAXX were identified using PhosphoSite.org. ER+ cells were treated with kinase inhibitors for AURKA (alisertib), AURKB (barasertib), CK1 (CK-IN-1), or CK2 (CX-4945) and DAXX protein was detected by western blotting. BCSC survival was measured using mammosphere forming assay. Based on results from the kinase inhibitor screen, barasertib was selected for pre-clinical testing in mice. ER+ MCF-7 xenograft tumors were treated with vehicle or barasertib in vivo up to 120 days. Results: DAXX protein levels decreased in ER+ PDX tumors in response to ET. Similar trends were detected in human tumor tissue after ET or chemotherapy alone. DAXX protein levels inversely correlated with Ki67 in ER+ human tumor samples. Analysis of DAXX deletion mutants demonstrated that amino acids 400-740 of the DAXX protein were required to inhibit BCSC survival. This region of the DAXX protein had high levels of phosphorylated residues in response to ET. Only the AURKB (barasertib) inhibitor increased DAXX protein expression and inhibited BCSCs in a DAXX-dependent manner. Barasertib inhibited growth of ER+ MCF-7 tumor xenografts compared to the vehicle control. After treatment ceased, tumor recurrence was measured up to 120 days. Regrowth of tumors was partially delayed in response to barasertib. Conclusions: ET decreased DAXX protein levels in ER+ PDX and human tumors. Downregulation of the DAXX protein by ET was through activation of AURKB and hyper-phosphorylation of DAXX which resulted in protein degradation and enhanced survival of BCSCs. Therefore, Inhibition of AURKB using barasertib partially restored DAXX expression, inhibited BCSCs, and delayed tumor recurrence. A combination approach of ET plus an AURKB inhibitor might be a novel therapeutic strategy to prevent ER+ breast cancer relapse by increasing DAXX in order to eradicate BCSCs. Support: Breast Cancer Research Foundation Citation Format: Clodia Osipo, Debra Wyatt, Kathy Albain. Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-12.

Abstract PO3-18-04: MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer

Abstract Objectives: ADCs have emerged as a major advance in contemporary medical oncology. The receptor tyrosine kinase (RTK) EphA5 possesses the classical receptor kinase topology with an extracellular-binding domain, a single-pass transmembrane domain, and a cytoplasmic kinase domain. We have previously shown that EphA5 is highly expressed in non-small cell lung cancer and contributes to DNA-damage repair and radiation therapy resistance. Others have reported EphA5 expression in gastric, ovarian, and pancreatic cancers. Here, we investigated the expression of EphA5 in breast cancer (BC) and assessed EphA5 as a potential cancer-specific target using a novel ADC designated MBRC-101. Methods: MBRC-101 is composed of a humanized anti-EphA5 IgG1 kappa monoclonal antibody conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease cleavable valine-citrulline (vc) ThioBridge linker (ThioBridge™-Glu-[Val-Cit-PAB-MMAE] PEG [24u]). MBRC-101 averages a drug-to-antibody ratio (DAR)=4. The specificity profile of the anti-EphA5 monoclonal antibody was characterized using a Membrane Proteome Array (MPA). Receptor-mediated antibody internalization was determined by real-time live-cell analysis; cell killing assays used both live-cell analysis of cell confluence and the CellTiter-Glo® luminescent cell viability assay. Expression of EphA5 in archival human breast tumor tissue sections was evaluated by immunohistochemistry (IHC) using a commercial polyclonal antibody against human EphA5. In vivo efficacy studies used patient-derived xenograft (PDX) models of BC. Results: IHC demonstrated robust and selective EphA5 expression in 87% (20 of 23) of triple negative breast cancer (TNBC) and 88% (23 of 26) of hormone receptor-positive (HR+) patient tumors tested. EphA5 expression was not detected in adjacent, non-malignant breast tissue. Antibody-specificity profile testing showed that the anti-EphA5 antibody bound exclusively to EphA5 and did not cross-react with other members of the Eph receptor/ephrin ligand family. Binding of the anti-EphA5 antibody to EphA5 on the cell surface triggered rapid internalization and processing of the EphA5/antibody complex through the endosomal-lysosomal system. Consistently, MBRC-101 was cytotoxic to EphA5-expressing cells and its activity was concentration-dependent and commensurate to the levels of EphA5 on the cell surface. In PDX murine models of TNBC, once weekly administrations of intravenous (IV) MBRC-101 showed dose-dependent, robust, and reproducible anti-tumor activity in vivo. Partial tumor responses were observed at doses of 2.5 mg/Kg IV and complete and nearly complete tumor responses starting at 5 mg/Kg IV. Doses up to 10 mg/Kg IV were well-tolerated in tumor-bearing mice with no observable weight loss. Pre-clinical safety confirmed MBRC-101 is also well tolerated in rats and in cynomolgus monkeys. Conclusions: These results support EphA5 as a new and promising membrane-associated molecular target for the design and development of ADC-based therapies against hormone-positive breast cancer and TNBC and support the conduct of a Phase 1/1b study of MBRC-101 in patients with breast cancer. Citation Format: Fernanda Staquicini, Fenny Tang, Vanessa de Oliveira, Daniela Staquicini, Yongjian Wu, Kirstin Barnhart, J Kellogg Parsons, Wadih Arap, Isan Chen, Renata Pasqualini. MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-04.

Multiplicative Improved Coherence Factor Delay Multiply and Sum Algorithm for Clutter Removal in a Microwave Breast Tumor Imaging System

In the medical field, microwave imaging technology has experienced rapid development due to its non-invasive and non-radioactive nature. The confocal algorithm is a method commonly used for microwave breast cancer imaging, with the key objective of removing clutter in images to achieve high-quality results. However, the current methods are facing challenges in removing clutter. In order to reduce the clutter in images, a multiplicative improved coherence factor delay multiply and sum algorithm based on the maximum interclass differencing method is proposed. The algorithm compares the starting and ending moments of tumor signals in different channels to determine whether the tumor-scattered signals in different channels overlap in time. An improved coherence coefficient is obtained by summing the non-overlapping signals and multiplying the time window. The multiplicative improved coherence factor, which is obtained by multiplying the coherence coefficients of the improved multi-pair signals, is then multiplied by the focal point intensity obtained using the delay multiply and sum algorithm to reduce clutter in an image. To evaluate the performance of the proposed algorithm, several low-cost uniform and non-uniform models of human breast and tumor tissue with dielectric properties were prepared for testing. The experimental results show that, compared to the existing algorithm, the proposed algorithm can greatly reduce the clutter in images, with a signal-to-clutter ratio of at least 4 dB higher as well as contrast at least six-fold higher.

Quantitative Assessment of Preanalytic Variables on Clinical Evaluation of PI3/AKT/mTOR Signaling Activity in Diffuse Glioma

Biomarker-driven therapeutic clinical trials require the implementation of standardized, evidence-based practices for sample collection. In diffuse glioma, phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR (PI3/AKT/mTOR) signaling is an attractive therapeutic target for which window-of-opportunity clinical trials could facilitate the identification of promising new agents. Yet, the relevant preanalytic variables and optimal tumor sampling methods necessary to measure pathway activity are unknown. To address this, we used a murine model for isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) and human tumor tissue, including IDH-wildtype GBM and IDH-mutant diffuse glioma. First, we determined the impact of delayed time-to-formalin fixation, or cold ischemia time (CIT), on the quantitative assessment of cellular expression of 6 phosphoproteins that are readouts of PI3K/AK/mTOR activity (phosphorylated-proline-rich Akt substrate of 40 kDa (p-PRAS40, T246), -mechanistic target of rapamycin (p-mTOR; S2448); -AKT (p-AKT, S473); -ribosomal protein S6 (p-RPS6, S240/244 and S235/236), and -eukaryotic initiation factor 4E–binding protein 1 (p-4EBP1, T37/46). With CITs ≥ 2 hours, typical of routine clinical handling, all had reduced or altered expression with p-RPS6 (S240/244) exhibiting relatively greater stability. A similar pattern was observed using patient tumor samples from the operating room with p-4EBP1 more sensitive to delayed fixation than p-RPS6 (S240/244). Many clinical trials utilize unstained slides for biomarker evaluation. Thus, we evaluated the impact of slide storage conditions on the detection of p-RPS6 (S240/244), p-4EBP1, and p-AKT. After 5 months, storage at −80°C was required to preserve the expression of p-4EBP1 and p-AKT, whereas p-RPS6 (240/244) expression was not stable regardless of storage temperature. Biomarker heterogeneity impacts optimal tumor sampling. Quantification of p-RPS6 (240/244) expression in multiple regionally distinct human tumor samples from 8 patients revealed significant intratumoral heterogeneity. Thus, the accurate assessment of PI3K/AKT/mTOR signaling in diffuse glioma must overcome intratumoral heterogeneity and multiple preanalytic factors, including time-to-formalin fixation, slide storage conditions, and phosphoprotein of interest.

Abstract LB193: Enhanced single cell DNA methylation analysis using combinatorial indexing

Abstract The epigenetic landscape of the human brain undergoes a plethora of changes during malignant transformation. In recent years, DNA methylation-based epigenetic modifications have been widely studied using traditional techniques like bisulfite sequencing and enzymatic methyl sequencing (EM-seq). However, these methods analyze bulk cell populations and lack the granularity of single-cell analysis. Although advances in single-cell analysis have revolutionized our understanding of cellular heterogeneity and functional diversity within complex biological systems, they are still costly, low throughput, and laborious. To address these challenges, ScaleBio has pioneered combinatorial indexing technology, enabling a significant increase in cell throughput. This method utilizes the cell itself as a compartment to perform 2-3 rounds of sequential barcoding in a plate-based workflow, eliminating the need for complex instrumentation. This technology has been successfully adapted to assess DNA methylation at the single-cell level offering a robust, affordable, high-throughput protocol that enhances yield, diversity, and coverage. In this study we used ScaleBio's single-cell methylation kit to investigate DNA methylation patterns during oncogenesis using cancer cells with widespread DNA methylation changes, such as human isocitrate dehydrogenase (IDH) mutant glioma cells at the single-cell level. The IDH gene family, comprising of IDH1, IDH2, and IDH3, encodes enzymes involved in the tricarboxylic acid (TCA) cycle. IDH1/2 mutations are present in over 80% of low-grade gliomas while IDH-mutant gliomas constitute about 1/5th of all adult diffuse gliomas, thus making them one of the most common brain tumor subtypes. As such, IDH mutations have emerged as attractive therapeutic targets for glioma treatment. By uncovering DNA methylation patterns at the single cell level, we provide here an epigenetic map to better understand this complex disease and better inform clinical discoveries. We achieved high cell recovery and robust cytosine coverage throughout our analysis of single cell methylomes isolated from human glioma tumor tissue. Using this data we generated a ranked list of the top hypo- and hypermethylated genomic regions and identified cell type specific clusters seen in different pathological states by looking at Differentially Methylated Regions (DMR) uncovering unique single-cell methylation profiles that may be obscured by bulk or pseudo-bulk analysis. These data show that the ScaleBio single-cell methylation workflow offers increased sensitivity, specificity, and accuracy in identifying DNA methylation sites when compared to other techniques while offering a comprehensive view of methylomes and providing insights into cellular heterogeneity and trajectories. Citation Format: Sanika Khare, Dominic Skinner, Maggie Nakamoto, Hosu Sin, Ashley Woodfin, Eric Pu, Lin Lin, Jason Koth, Beth Walczak. Enhanced single cell DNA methylation analysis using combinatorial indexing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB193.

Abstract LB049: Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway

Abstract Lung cancer is the second most common cancer in both men and women in the United States. Even though treatments including chemotherapy, targeted therapy, and immunotherapy are available, lung cancer has been the leading cause of cancer deaths. The discovery and evaluation of new alternative medications targeting lung cancer are tremendously important for reducing lung cancer mortality. Phytochemicals are very desirable chemotherapeutic agents because of their greater safety profile. Isorhapontigenin (ISO), one of the oral bioavailable dietary stilbenes, can be found in various natural resources. Our result showed that ISO treatment suppressed the invasion and migration of human lung cancer cell lines A549, H23, and H1299 cells. Long non-coding RNA Maternally Expressed Gene 3 (MEG3), a tumor suppressor, was downregulated or lost in various primary human tumor tissues and cancer cell lines. The low expression of MEG3 is correlated with poor prognosis in non-small cell lung cancer (NSCLC). Our results showed that ISO treatment elevated MEG3 levels in these lung cancer cell lines. One of the major genetic causes of lung cancer is mutations in the epidermal growth factor receptor (EGFR) receptor tyrosine kinase, resulting in activation of EGFR which activates several signaling pathways involved in cell proliferation, drug sensitivity, and angiogenesis. Our results showed that ISO decreased the phosphorylation and protein levels of EGFR in A549 cells. Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, regulates the activation of several genes involved in cell proliferation, angiogenesis, invasion, and metastasis. Our results showed that ISO decreased HIF-1α protein levels. Overexpression of MEG3 reduced p-EGFR and HIF-1α, indicating MEG3 negatively regulated EGFR and HIF-1α. Our data also showed that inhibition of EGFR by its tyrosine inhibitor decreased HIF-1α, suggesting that EGFR positively regulates HIF-1α. In cancer cells, Epithelial-Mesenchymal Transition(EMT) endows cells with increased invasiveness and tumor-initiating capacity, contributing to metastasis and resistance to therapeutics. Slug, one of the EMT transcription factors maintains cell plasticity during carcinogenesis. Our results showed that ISO treatment decreased Slugprotein levels in A549 and H23 cells. Transcription factor SOX2 regulates cancer cell proliferation, EMT, migration, invasion, metastasis, tumor initiation, cancer stem cell formation as well as resistance to apoptosis and therapy. Our results showed that ISO decreased SOX2 expression. Knockdown of MEG3 increased the expressions of Slug and SOX2, indicating that MEG3 negatively regulates Slug and SOX2. In conclusion, ISO elevated MEG3, inhibiting EGFR, HIF-1α, Slug, and SOX2, suppressing the migration and invasion of lung cancer cells. Citation Format: Sophia Shi, Zhuo Zhang, Jingxia Li, Huailu Tu, Max Costa. Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB049.

The mechanism of extracellular CypB promotes glioblastoma adaptation to glutamine deprivation microenvironment

Glioblastoma, previously known as glioblastoma multiform (GBM), is a type of glioma with a high degree of malignancy and rapid growth rate. It is highly dependent on glutamine (Gln) metabolism during proliferation and lags in neoangiogenesis, leading to extensive Gln depletion in the core region of GBM. Gln-derived glutamate is used to synthesize the antioxidant Glutathione (GSH). We demonstrated that GSH levels are also reduced in Gln deficiency, leading to increased reactive oxygen species (ROS) levels. The ROS production induces endoplasmic reticulum (ER) stress, and the proteins in the ER are secreted into the extracellular medium. We collected GBM cell supernatants cultured with or without Gln medium; the core and peripheral regions of human GBM tumor tissues. Proteomic analysis was used to screen out the target-secreted protein CypB. We demonstrated that the extracellular CypB expression is associated with Gln deprivation. Then, we verified that GBM can promote the glycolytic pathway by activating HIF-1α to upregulate the expression of GLUT1 and LDHA. Meanwhile, the DRP1 was activated, increasing mitochondrial fission, thus inhibiting mitochondrial function. To explore the specific mechanism of its regulation, we constructed a si-CD147 knockout model and added human recombinant CypB protein to verify that extracellular CypB influenced the expression of downstream p-AKT through its cell membrane receptor CD147 binding. Moreover, we confirmed that p-AKT could upregulate HIF-1α and DRP1. Finally, we observed that extracellular CypB can bind to the CD147 receptor, activate p-AKT, upregulate HIF-1α and DRP1 in order to promote glycolysis while inhibiting mitochondrial function to adapt to the Gln-deprived microenvironment.

THE ROLE OF piRNAs IN ONCOGENESIS: A REVIEW OF ASSOCIATIONS

Background. The cumulation of data on molecular genetic events associated with oncogenesis resulted into transformation of the understanding of the role of small non-coding RNAs in the process. The largest class of these molecules is represented by piRNAs. In recent years, there has been a significant increase in the number of experimental studies uncovering functions of piRNAs, in particular, on their role in tumor cell transformation. This knowledge can be applied both in fundamental and practical oncology. Aim. To summarize data from published scientific papers on the association of piRNAs with human malignancies. Material and methods. The review represents analysis of studies on the effect of piRNAs on oncogenesis in humans. PubMed and eLIBRARY.RU databases were searched using the combinations of the terms («Carcinogenesis/Oncogenesis» OR «Cancer/Tumor») AND «piRNA», and the «Humans» as filter. Search was limited by the publication date until 01 January 2008. Results. 25 studies were included in the review. Among them with the aid of various molecular genetic methods ectopic levels of endogenous piRNAs expression were demonstrated in human tumor tissues or cell cultures, of which: for 36 piRNAs – increased, for 19 piRNAs – decreased, and for the piR-823 and piR-651 – aberrant multidirectional depending on one of nine tumor types. Modulation of piRNAs expression levels in many tumors was associated with changes in cell proliferation, apoptosis,tumor growth and metastasis, sensitivity to chemotherapeutic treatments, and other effects.Conclusion. Non-coding piRNAs play an important role in oncogenesis, determination of their level in tumors may have diagnostic or prognostic significance and modulation of the level of their expression may represent an effective antitumor therapeutic strategy

Label-free quantification of imaging features in the extracellular matrix of left and right-sided colon cancer tissues

The molecular pathogenesis of colorectal cancer is known to differ between the right and left side of the colon. Several previous studies have focussed on the differences in clinicopathological features, proteomic and genetic biomarkers, the composition of gut microbiota, response to therapy, and the characteristics of the tumour microenvironment. However, the morphology and density of collagen in the extracellular matrix (ECM) have not been studied intensively. In this study, we employed 2-photon laser scanning microscopy (2PLSM) to visualise the intrinsic second-harmonic generation (SHG) signal emitted by collagen fibres in the heterogeneous ECM of human colon tumour tissues. Through texture analysis of the SHG signal, we quantitatively distinguished the imaging features generated by structural differences of collagen fibres in healthy colon and cancers and found marked differences. The fibres inside of tumours exhibited a loss of organisation, particularly pronounced in right-sided colon cancer (RSCC), where the chaotic regions were significantly increased. In addition, a higher collagen content was found in left-sided colon cancer (LSCC). In future, this might aid in subclassification and therapeutic decisions or even in designing new therapy regimens by taking into account the differences between collagen fibres features between colon tumours located at different sides.

Abstract 7166: Pharmacokinetic analysis of omomyc therapeutic protein in preclinical and human tumor tissue using targeted mass spectrometry

Abstract MYC is a pleiotropic transcription factor consisting in three paralogs which coordinate gene expression and regulates cell proliferation, cell differentiation, cell cycle, metabolism and apoptosis. The 3 paralogs (c-, N-, and L-MYC) have similar biological functions but show distinct tissular and timing expression patterns. To function, MYC must heterodimerize with its partner MAX and bind DNA via its basic, helix-loop-helix, leucine zipper domain (B-HLH-LZ). It has been estimated that MYC is aberrantly expressed in up to 70% of human cancers and hence MYC has been termed a “most wanted target for cancer treatment”. However, due to its nuclear localization, its intrinsic disorder and lack of an enzymatically active site, development of clinical grade potent & selective small molecules targeting MYC remains an obstacle. Omomyc is a 91-amino-acids-long polypeptide that derives from the B-HLH-LZ domain of human MYC, containing four amino acid substitutions. Omomyc is capable of forming homodimers and heterodimers with MYC and with MAX and such dimers blunt MYC activity by preventing its access to target DNA sequences (E-boxes) and replacing it by inactive complexes. Previous studies showed the cell-penetrating properties of recombinant Omomyc and its subsequent nuclear localization, where it displaces MYC from its genomic locations, causing a specific shutdown of MYC transcriptional signature and effectively stopping cell cycle progression. Further in vivo preclinical experiments showed that Omomyc blocks MYC-driven transcription and proliferation, induces apoptosis and recruitment of intratumoral immune infiltrates, overall significantly abrogating tumor progression and reducing tumor grading. A targeted LC-MS/MS assay for the absolute quantification of 4 Omomyc proteotypic peptides was developed to assess the structural integrity of Omomyc and quantify functional Omomyc in tumor tissue. LLOQ and linear range of the panel was determined by spiking in dilution series of recombinant Omomyc protein into preclinical tumor tissue lysates. Finally, in the context of Omomyc clinical trial (NCT04808362), we used the panel to for quantification of functional Omomyc in FFPE tumor biopsies from 18 patients who received i.v. injection of Omomyc therapeutic. Data show that 2 h after i.v. administration, higher concentrations of the drug are reached in the tumors compared with serum and persist there with at least one order of magnitude higher concentration than in serum 72 h after administration in preclinical models. Functional Omomyc protein could be quantified in all of the 18 clinical tumor biopsies from treated patients showing that characterized LC-MS/MS proteomic assay can be used in the normal routine of a clinical trial for accurate quantification of therapeutic miniproteins. Citation Format: Marie-Eve Beaulieu, Sandra Martinez-Martin, Maik Mueller, Yuehan Feng, Jakob Vowinckel, Antoine Amaury Lachaud, Laura Soucek. Pharmacokinetic analysis of omomyc therapeutic protein in preclinical and human tumor tissue using targeted mass spectrometry [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 7166.

Abstract 1044: Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™

Abstract Background: Multiplex immunofluorescence (mIF) is an important tool for extracting complex phenotypic information from normal or diseased tissues, and provides accurate and predictive insights on the tissue microenvironment faster. For patient samples in translational and clinical research, high sensitivity, specificity, and reproducibility are critical; however, throughput and cost considerations are also important in designing clinical studies. Enabling an easy-to-use high-performance assay such as InSituPlex® on robust and affordable auto-stainers like the Parhelia Omni-Stainer™ can allow researchers to gain a deeper understanding of the tissue micro-environment through high-quality multiplexed biomarker detection in a fraction of the time and cost. Methods: We performed Ultivue InSituPlex® assays with OmniVUE™ panels using the Parhelia Omni-Stainer™, a low-cost, high-throughput tissue auto-stainer with reduced requirements for reagents with a minimal dead volume. We compared the whole-slide staining performance across replicates using human tonsil and tumor tissue arrays across multiple independent runs over multiple days. The stained slides were scanned using the Zeiss AxioScan.Z1 high-throughput imaging system, and image batches were processed using Ultivue’s UltiStacker.AI and UltiAnalyzer.AI software. Results: Tighter temperature control of the slide in the Omni-Stainer supports optimal InSituPlex® signal amplification. Intra-run and inter-day analysis on serial sections of human tonsil and tumor tissue arrays demonstrated a high degree of consistency in InSituPlex® assay performance, specifically with marker-specific signal intensities and cell densities. The Omni-Stainer facilitates the use of low reagent volumes at each step of the assay due to its efficient reagent use, thus lowering the cost of reagents per slide. Conclusions: This study demonstrated a fast, simple and robust workflow to perform high-performance InSituPlex® mIF assays on the low-cost, high-throughput Parhelia Omni-Stainer™. Ultivue’s configurable OmniVUE™ mIF panels can be run on the Omni-Stainer with reduced associated reagent costs per slide. This workflow can support larger clinical and translational studies in a more cost-effective fashion, leading to in-depth analysis of tissue microenvironment through highly precise multiplexed biomarker detection. Citation Format: Kevin Hwang, Lauren Duro, Alex Veith, Nelson Wedin, Nikolay Samusik, Je H. Lee. Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™ [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 1044.

Abstract 4200: A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells

Abstract Human cancer tissue is a complex ecosystem with heterogeneous cell types that communicate with each other dynamically to promote tumor progression and immune evasion. Hence, experimental systems that destroy or select for cancer cells in culture have often failed to accurately predict clinical responses of new drugs. Therefore, a technology that can retain the intact tumor microenvironment (TME) and accurately predict cancer patient’s response to anti-cancer treatments, including immunotherapies, will greatly benefit patients and dramatically reduce the cost of new drug development. E-slice is a proprietary 3D human tumor culture platform that maintains individual patient tumor’s unique ecosystem and the TME ex vivo for up to 30 days. It is generated by making thin sections of intact, fresh tumor tissues and culturing them in serum-free, defined media. As such, immune components and the TME in E-slices faithfully recapitulate human tumors ex vivo. We demonstrate that E-slices can be used to: 1) measure viability changes upon anti-cancer agent treatment longitudinally; 2) retain the native TME and tissue architecture since E-slices are never dissociated or artificially reconstituted; 3) culture any solid tumor types from patient needle biopsy cores or surgical samples, PDX, or mouse models; 4) perform high content drug screening on human tissues; 5) discover secreted biomarkers and to 6) discover or validate molecular mechanisms of action or therapy resistance; and 7) measure immunotherapy responses ex vivo from human tissues. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-8 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. Furthermore, single cell RNA-sequencing analysis demonstrates that E-slices can retain the viability and molecular phenotypes of tumor infiltrating immune cells for up to 8 days ex vivo, providing a unique platform to study human tumor-immune interactions and evaluate efficacy of immune modulators on human tumor tissues ex vivo. Citation Format: Viridiana Leyva-Aranda, Thomas Gallup, Jose Maldonado, Corina Margain, Sang Yun, David Gallup, Min Kim, Kyuson Yun. A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells [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 4200.

Abstract 2721: FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity

Abstract Background: Human endogenous retroviruses (HERVs) are transposable elements (TE) derived from retrovirus infections that occurred millions of years ago. Despite constituting 8% of our genome, their biological role still needs to be explored. HERV-K (HML-2), a subgroup of HERVs, represents the most recent retroviruses integrated into the human germline DNA. Members of the HML-2 subgroup retain complete ORF with coding potential and have garnered the most attention in disease-related studies. HERVs exhibit physiological expression in embryonic stem cells but are heavily methylated and remain almost silent in normal adult tissues. Although the mechanisms underlying HERV-K contribution to tumor cell growth and survival remain poorly understood, aberrant expression of HERVs has been demonstrated in various human tumor tissues and lymphoma. These findings led us to explore HERV-K as a potential therapeutic target in B cell lymphomas .Methods: RNA data were analyzed with Telescope. Validations were performed by qPCR, Western blot, flow cytometry, and ELISA. Env structure was obtained with AlphaFold and peptides were purchased from GeneScript. Antibody-dependent cellular cytotoxicity (ADCC) was performed with the ADCC Reporter Bioassay (Promega). Internalization was assessed using the Incucyte Fabfluor-pH Antibody labeling reagents. Results: We assessed the TE expression in RNA-seq datasets of diffuse large B cell lymphoma (DLBCL) and B cells from healthy individuals. An MDS plot showed that TEs could separate neoplastic from healthy samples, demonstrating that tumoral and normal specimens have distinct TE and HERV expression profiles. Focusing on tumor samples, DLBCLs were divided into four clusters with different prognoses. Similar results were obtained in a 47 B-cell lymphoma cell line panel. Focusing on the HML-2 family in lymphoma patients, we identified many of them upregulated in tumors. We validated RNA and protein expression of the HML-2 envelope (env) in seventeen lymphoma cell lines, and in two of them, we also proved the protein surface expression. Env-specific siRNA reduced env expression determined a decrease in cell proliferation, suggesting a contribution to lymphoma cell growth. In silico examination of the env identified four linear regions with potential for antibody binding, and peptides covering them were synthesized. We screened 150 lymphoma patients' serum samples for IgG antibodies binding to the peptides, and all peptides showed positive hits. We used a phage display screen to obtain one nanobody, FF01, binding one peptide. FF01 induced ADCC in the cell line with high surface env expression and not in a negative one. FF01 was internalized in an expressing cell, proving the principle for using it as a component of an antibody-drug conjugate. Conclusions: Our data demonstrate that HERVs are in DLBCL patients and propose FF01 as a new candidate to target HERV Env+ lymphomas. Citation Format: Filippo Spriano, Luciano Cascione, Jacopo Sgrignani, Nikolai Bendik, Sara Napoli, Giulio Sartori, Eleonora Cannas, Tao Gongo, Alberto J. Arribas, Davide Rossi, Davide F. Robbiani, Andrea Cavalli, Francesco Bertoni. FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity [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 2721.

Abstract 3797: Unveiling the spatial dynamics of the tumor microenvironment: Integrated RNA and protein profiling on the same slide through automated spatial multiomics analysis

Abstract Spatial biology techniques have revolutionized our approach towards the study of the tumor microenvironment (TME) and its complex cellular interplay. On the one hand, multiplex immunofluorescence (mIF) methods have enabled a precise profiling of immune cells and other key cellular players of the TME while uncovering their spatial distribution and interactions. On the other hand, in situ hybridization (ISH) technologies have shown to provide complementary information to protein profiling such as the mapping of cytokine- and chemokine-expressing cells, essential for comprehending signaling networks and immune activation statuses. Here, we made use of a novel multiomics approach that combines these two biological inputs by integrating RNAscope™ and sequential immunofluorescence (seqIF™) protocols to achieve same-slide co-detection of RNA and protein targets. The combined workflow is automated on COMET™, an advanced tissue staining and imaging platform. Through precise control over temperature and reagent distribution, the instrument ensures maximum efficiency and reproducibility of the assays. The integrated multiomics protocol allows for up to three RNAscope™ detection cycles combined with twelve seqIF™ cycles, for a final 12-plex RNA + 24-plex protein panel. We first showed the capacity of COMET™ platform to fully automate the RNAscope™ protocol and demonstrated its sensitivity and specificity with the analysis of positive and negative control genes on HeLa cell pellets, highlighting the flexibility of sample selection. To illustrate the potential multiomics in unravelling the complexity of the TME, we designed a panel of 12-RNA-targeting probes for key biomarkers of tumor-infiltrating lymphocytes and their activation status, including multiple secreted molecules (e.g., cytokines and proteases). This was combined with a 24-antibody panel for the detection of protein biomarkers, selected to enable the single-cell profiling of different players within the TME. We applied this panel to characterize different human FFPE tumor tissues and showed that the co-detection of RNA and protein biomarkers on the same slide allows a better characterization of key cellular components involved in tumor progression and immune response. Our results highlight the potential of spatial multiomics technologies in enhancing the efficiency of investigations of immune cell behavior and generally the understanding of cellular interplay in the TME. Their full automation on a platform like COMET™ will accelerate analysis and increase robustness by minimizing user intervention, and ultimately helping in the development of prognostic and predictive biomarkers, in the refinement of cancer diagnoses, and in the selection of new personalized therapies. Citation Format: Paula Juricic, Arec Manoukian, Alice Comberlato, Pino Bordignon, Alix Failletaz, Anushka Dikshit, Emerald Doolittle, Rose Delvillar, Steve Zhou, Ching-wei Chang, Li-chong Wang, Saska Brajkovic, Maithreyan Srinivasan, Diego Dupouy. Unveiling the spatial dynamics of the tumor microenvironment: Integrated RNA and protein profiling on the same slide through automated spatial multiomics analysis [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 3797.

Abstract 5516: Aberrant activation and perpetuation of lung wound response facilitates osteosarcoma metastasis

Abstract Introduction: The mechanisms that allow tumor cells to integrate with unfamiliar parenchymal cells within hostile distant tissues during metastatic colonization remain poorly defined. Nowhere are these mechanisms more important than in osteosarcoma-a pediatric bone tumor where metastases cause nearly all osteosarcoma related deaths and occur almost exclusively in the lung. The purpose of this study was to define the cellular and molecular events that facilitate osteosarcoma lung colonization to devise novel therapeutic strategies that prevent and treat metastasis. Experimental Approach: We generated experimental lung metastases using a panel of immunocompetent murine and human xenograft osteosarcoma models. A combination of immunohistochemistry, single cell RNA sequencing, and spatial transcriptomics were used to define the metastatic niche. Relevant findings were validated in human primary tumor, lung metastasis, and normal lung tissue specimens. Results: Disseminated osteosarcoma cells physically associate with alveolar epithelial cells and trigger changes in surrounding epithelial cells that are characteristic of the aberrant, non-resolving wound response seen in non-malignant lung diseases such as idiopathic pulmonary fibrosis (IPF). This process drives the accumulation of fibrotic epithelial cells and scar-associated macrophages confined within a dense fibronectin-based matrix. Anti-fibrotic tyrosine kinase inhibitors used to treat patients with IPF (nintedanib) prevent the observed fibrogenic changes and inhibit metastatic colonization. Epithelial-osteosarcoma paracrine signaling is dominated by an inflammatory cytokine milieu that facilitates this transition towards fibrosis. Blocking one of the dominant paracrine signals with a recombinant IL1 receptor antagonist (anakinra) has similar effects on both fibrosis and metastatic colonization. Conclusions: Our study demonstrates that tumor interactions with host stromal cells are critical for metastasis. Metastatic colonization requires transformation of the local lung microenvironment into a fibrotic, non-resolving, cytokine-rich, wound-like state that is both provoked and sustained by tumor-epithelial interactions. These insights have identified several potential therapeutic approaches to prevent metastatic colonization and treat established metastases. Citation Format: James Brandon Reinecke, Amy C. Gross, Maren Cam, Leyre Jimenez Garcia, Matthew V. Cannon, Ryan D. Roberts. Aberrant activation and perpetuation of lung wound response facilitates osteosarcoma metastasis [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 5516.

Abstract 237: Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and there is a critical need for more effective treatments. The development of new therapies, especially those aiming to modulate the tumor microenvironment (TME), is limited by the lack of in vitro and in vivo models that recapitulate the complexity of the TME and spatial architecture of HCC. Here, we aimed to establish an HCC model to assess the treatment response and hepatotoxicity of selected drugs by ex vivo culturing fresh human liver tumor tissue and adjacent normal tissue, respectively. Materials and Methods: Fresh human liver tissues, including 5 malignant and 20 adjacent-normal, were obtained from surgical resections. PDX tumors were generated from patient-derived organoids. Tissue resections were cut into ultra-thin slices (from 150µm to 350µm) using a vibratome. Related culture conditions such as high oxygen partial pressure, air-liquid interface (ALI) culture, artificial extracellular matrix (AEM), and delay duration were evaluated to optimize the protocol. As a readout, cell viability was quantified by an expert pathologist using Hematoxylin/Eosin staining. The preservation and complexity of the TME were evaluated by immunohistochemistry or flow cytometry using specific antibodies for each cell population (T cells, anti-CD3; stroma cells, a-SMA; endothelial cells, CD34; Kupffer cells, CD68). Liver cell function was tested by measuring ALT/AST activity, albumin release, and CYP340 activity. Results: The highest tissue viability was observed in normal and tumor tissue with a thickness of 150µm and 250µm, respectively. Compared to standard conditions, the combination of ALI and high-oxygen partial pressure conditions dramatically improved tissue viability, with most of the samples having 70% and 50% cell viability at days 3 and 5 of ex vivo culture, respectively (p<0.05). AEM could prevent the efflux of cellular components (p=0.016). The impact of the time delay between the liver resection and the start of slicing was minimal when the slicing occurred within two hours after hepatectomy (p<0.05). Key cellular components including hepatocytes, T cells, fibroblasts, and Kupffer cells also remained better preserved throughout the 5-day culture period in optimized conditions. Conclusion: Our study has successfully optimized the ex vivo tissue culture model of liver tissue, capable of maintaining the tissue for several days while preserving the unique TME. These results highlight the potential of using the ex vivo tissue culture model in the preclinical setting to further investigate and evaluate TME-related treatments and their hepatotoxicity in HCC tissues. Citation Format: Huiche Feng, Miriam Cieri, Cinzia Esposito, Karolina Guja-Jarosz, Daniela Liberati, Lukas Bubendorf, Savas Deniz Soysal, Mairene Coto-Llerena, Luigi Maria Terracciano, Otto Kollmar, Salvatore Piscuoglio. Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma [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 237.