Conditions In Tumor Microenvironment Research Articles

VEGF-A as a prognostic indicator in advanced gastric and colorectal cancers and correlation with immune suppression involving myeloid-derived suppressor cells (MDSC).

250 Background: VEGF-A is important molecule should be controlled in cancer treatment since hypoxic and immunosuppressive conditions of tumor microenvironment are induced by VEGF-A. Myeloid-derived suppressor cells (MDSC), activated by chronic inflammation and VEGF-A, play critical roles in immunosuppression in patients with cancer. Methods: Serum levels of VEGF-A were measured by ELISA and circulating levels of MDSC were measured by Flow Cytometry (CD11b+, CD14-, CD33+). Patients with gastric and colorectal cancers were divided into two groups, high VEGF group (serum VEGF-A>330pg/ml) and lo group (≤330pg/ml). Results: Prognosis of stages III and IV gastric cancer with high VEGF-A was significantly worse than in low VEGF-A group, while difference was not significant in stages I and II. In colorectal cancer, the results were exactly same as found in gastric cancer. The levels of MDSC were significantly correlated with the levels of VEGF-A in patients with gastric and colorectal cancers. The levels of VEGF-A were also correlated with inflammatory markers including CRP and NLR (neutrophil to lymphocyte ratio), and inversely did to stimulation index of lymphocyte proliferation (SI: parameter of cellular immune reaction) and nutritional parameters such as albumin or rapid turnover protein. Conclusions: The observation in the present study suggest that VEGF-A is closely associated with inflammation-associated disease progression especially in advanced stages, and serve as useful marker of immunosuppression involving MDSC and prognosis in patients with gastric and colorectal cancers.

A tumor microenvironment-responsive multifunctional MoS2-Ru nanocatalyst with photothermally enhanced chemodynamic activity.

Targeting the unique characteristics of the tumor microenvironment (TME) has emerged as a highly promising strategy for cancer therapy. Chemodynamic therapy (CDT), which leverages the TME's intrinsic properties to convert H2O2 into cytotoxic hydroxyl radicals (˙OH), has attracted significant attention. However, the effectiveness of CDT is often limited by the catalytic efficiency of the materials used. Although Molybdenum disulfide (MoS2) exhibits remarkable chemodynamic and photothermal properties, its limited efficiency in catalyzing the conversion of endogenous H2O2 into ˙OH radicals remains a significant challenge. To overcome this, we developed a nanocomposite of MoS2 and ruthenium (MoS2-Ru), by incorporating Ru into MoS2 nanosheets. The MoS2-Ru nanocomposite demonstrated significantly enhanced catalytic activity at a low concentration (500 ng mL-1), whereas the same effect was achieved only with 20 μg mL-1 of MoS2. The low Michaelis-Menten constant (Km) of 4.69 mM further confirmed the superior catalytic activity of the nanocomposite, indicative of the enhanced enzyme-like activity. Additionally, the integration of Ru in MoS2 reduced the bandgap to 1.18 eV, facilitating near-infrared (NIR) absorption with a high conversion efficiency of 41%. Electron paramagnetic resonance (EPR) analysis confirmed robust ˙OH radical generation driven by the combined chemodynamic and photothermal effects. In vitro studies using triple-negative breast cancer (TNBC) cells validated the synergistic activity of CDT and PTT, demonstrating significant ˙OH radical production under TME conditions, leading to effective cancer cell death. This study underscores the potential of MoS2-Ru nanocomposites as a versatile and powerful platform for multimodal cancer therapy, seamlessly integrating CDT and PTT to achieve synergistic, precise, and highly effective treatment outcomes.

A TaqMan-Based qRT-PCR Assay for Accurate Evaluation of the Oncogenic TrkAIII Splice Variant in Tumor cDNAs

Background: Alternative NTRK1/TrkA splicing resulting in TrkAIII expression, originally discovered in advanced-stage metastatic neuroblastomas, is also pronounced in prostate, medullary thyroid, glioblastoma multiforme, MCPyV-positive Merkel cell, cutaneous malignant melanoma, and pituitary neuroendocrine tumor subsets. In tumor models, TrkAIII exhibits actionable oncogenic activity equivalent to the TrkT3-fused oncogene, and in tumor cell lines, alternative TrkAIII splicing is promoted by hypoxia, nutrient deprivation, endoplasmic reticulum stress, and SV40 large T antigen, implicating tumor microenvironmental conditions and oncogenic polyoma viruses in tumor-associated TrkAIII expression. Collectively, these observations characterize TrkAIII as a potentially frequent, actionable oncogenic alternative to TrkA gene fusion in different tumor types. Currently, therapeutic approval for efficacious Trk inhibitors is restricted to Trk-fused gene positive tumors and not for tumors potentially driven by TrkAIII. Methods: With the therapeutically relevant aim of improving the identification of tumors potentially driven by TrkAIII, we have developed a TaqMan-based qRT-PCR assay for evaluating TrkAIII expression in tumor cDNAs. Results: This assay, validated using gel-purified fs-TrkA and TrkAIII cDNAs alone and in complex cDNA mixtures, employs primers and probes designed from fs-TrkA and TrkAIII sequences, with specificity provided by a TaqMan probe spanning the TrkAIII exon 5–8 splice junction. It is highly efficient, reproducible, and specific and can detect as few as 10 TrkAIII copies in complex RNAs extracted from either fresh or FFPE tumor tissues. Conclusions: Inclusion of this assay into precision oncology algorithms, when paired with fs-TrkA qRT-PCR and TrkA immune histochemistry, will make it easier to identify patients with therapy-resistant, advanced-stage metastatic Trk-fused gene-negative tumors potentially driven by TrkAIII, for whom approval of third-line effective Trk inhibitors could be extended.

Ribosome biogenesis, altered metabolism and ribotoxic stress response in pancreatic ductal adenocarcinoma tumor microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a poor overall survival rate. Cellular stress response pathways promoting cancer cell fitness in harsh tumor microenvironment (TME) play a critical role in cancer growth and survival. The influence of oncogenic Kras, multi-functional heterogeneous cancer-associated fibroblasts (CAFs), and immunosuppressive TME on cancer cells makes the disease more complex and difficult to treat. The desmoplastic reaction by CAFs comprises approximately 90% of the tumor, with only 10% of cancer cells making things even more complicated, resulting in therapy resistance. Consistently increasing fibrosis creates a hypoxic environment and elevated interstitial fluid pressure inside the tumor constraining vascular supply. Stress conditions in TME alter translation efficiency and metabolism to fulfill the energy requirements of rapidly growing cancer cells. Extensive research has been conducted on multiple molecular and metabolic regulators in PDAC TME. However, the role of TME in influencing translation programs, a prerequisite for cell cycle progression and functional/growth requirements for cancer cells, remains elusive. This review highlights the recent advancements in understanding altered translational programs in PDAC TME. We emphasize the role of ribosome biogenesis, ribosome-induced stress response, and the concept of specialized ribosomes and their probable role in mutationally rewiring the pancreatic TME.

Tumor Microenvironment and Dermatological Conditions in Prostate Cancer

Tumor initiation, progression, and invasion are closely related to the tumor microenvironment. Inflammation can modulate the activity of the cells in the TME and contribute to all stages of tumor development. The etiopathogenesis of cutaneous manifestations associated with prostate cancer is unclear. The cutaneous phenotype associated with prostate cancer could be supported by intratumoral heterogeneity, the remodeling of interactions in the tumor microenvironment, and the dynamics of the epithelial–mesenchymal transition. Among the urinary system cancers, prostate cancer presents few cutaneous signs and symptoms, most being diagnosed in the advanced stages of the disease. In this review, we analyze the cutaneous events associated with prostate cancer, represented by direct or indirect manifestations of the primary malignancy and the skin toxicities caused by oncological medications.

CXC chemokine receptor 4 - mediated immune modulation and tumor microenvironment heterogeneity in gastric cancer: Utilizing multi-omics approaches to identify potential therapeutic targets.

G-protein-coupled receptors (GPRs) are critical regulators of various biological behaviors, and their role in gastric cancer (GC) progression is gaining increasing attention. Among them, the immune regulatory mechanisms mediated by chemokine receptor 4 (CXCR4) remain insufficiently understood. This study aims to explore the immune regulatory functions of CXCR4 and the heterogeneity of the tumor microenvironment (TME) by examining GPR-related gene expression in GC. Through multi-omics approaches, including spatial transcriptomics and single-cell RNA sequencing, we investigated the oncogenic mechanisms of CXCR4, particularly its role in T cell immune exhaustion. In vitro experiments, including ELISA, PCR, CCK8 assays, cell scratch assays, and colony formation assays, were used to validate the role of CXCR4 in the migration and invasion of AGS and SNU-1 cell lines. CXCR4 silencing using siRNA further demonstrated its regulatory effects on these cellular processes. Our results revealed a strong correlation between elevated CXCR4 expression and increased exhaustion of regulatory T cells (Tregs) in the TME. Furthermore, heightened CXCR4 expression was linked to increased TME heterogeneity, driven by oxidative stress and activation of the NF-κB pathway, promoting immune evasion and tumor progression. Silencing CXCR4 significantly inhibited the invasive and proliferative abilities of AGS and SNU-1 cells, while also reducing the expression of pro-inflammatory cytokines IL-1β and interleukin-6, thus alleviating chronic inflammation and improving TME conditions. In conclusion, our comprehensive investigation highlights CXCR4 as a key mediator of TME dynamics and immune modulation in GC. Targeting CXCR4 presents a promising therapeutic strategy to slow tumor progression by reducing Tregs-mediated immune exhaustion and TME heterogeneity, positioning it as a novel therapeutic target in GC treatment.

Deciphering tumour microenvironment and elucidating the origin of cancer cells in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (CCC) has an East Asian preponderance. It is associated with endometriosis, a benign condition where endometrial (inner lining of the uterus) tissue is found outside the uterus and on the peritoneal surface, in the abdominal or pelvic space. CCC is relatively more resistant to conventional chemotherapy compared to other ovarian cancer subtypes and is associated with a poorer prognosis. In this study, we recruited and obtained tumour tissues from seven patients across the four stages of CCC. The tumour and the tumour microenvironment (TME) from 7 CCC patients spanning clinical stages 1-4 were transcriptionally profiled using high-resolution scRNA-seq to gain insight into CCC's biological mechanisms. Firstly, we built a scRNA-seq resource for the CCC tumour microenvironment (TME). Secondly, we identified the different cell type proportions and found high levels of immune infiltration in CCC. Thirdly, since CCC is associated with endometriosis, we compared CCC with two publicly available endometriosis scRNA-seq datasets. The CCC malignant cells showed similarities with glandular secretory and ciliated epithelial cells found in endometriosis. Finally, we determined the differences in cell-cell communication between various cell types present in CCC TME and endometriosis conditions to gain insights into the transformations in CCC.

Precise Regulation of Reactive Oxygen Species in Tumor Microenvironment for Alleviating Inhibitory Immunogenic Cell Death.

High level of reactive oxygen species (ROS) within the tumor microenvironment (TME) not only damage tumor cells but also diminish the efficacy of immunogenic cell death (ICD) and the activity of tumor-infiltrating T lymphocytes, thereby limiting the effectiveness of immunotherapy. Therefore, precise modulation of ROS level is crucial to effectively eliminate tumor cells and activate ICD-induced immunotherapy. Here, an intelligent yolk shell nanoplatform (SPCCM) that features calcium carbonate shells capable of decomposing under acidic TME conditions, thereby releasing the natural antioxidant proanthocyanidins (PAs) and the photosensitizer Ce6 is designed. PAs scavenge ROS within tumors, extending the survival time of T lymphocytes, while Ce6, as an ICD inducer, generates high ROS concentrations upon laser irradiation, thus reaching the toxic threshold within tumor cells and inducing apoptosis. The resulting apoptotic cells serve as tumor-associated antigens, promoting dendritic cells (DCs) maturation, and activating ICD. By effectively neutralizing ROS in the TME, PAs sustainably reduce ROS level, thereby enhancing DCs activation and restoring antitumor immune cell activity suppressed by ROS (resulting in an eightfold increase in DCs activation). This study demonstrates effective synergistic effects between photodynamic therapy and immunotherapy by precisely modulating ROS level.

Estimating Impacts of p16 Status on Tumor Radiosensitivity in Head and Neck Cancer using Predictive Models.

The intrinsic radiosensitivity index (RSI) and genomic-adjusted radiation dose (GARD) were reported to be able to predict the surviving fraction at 2 Gy and therapeutic effect when delivering actual treatment doses using the gene expression profiles of clinical cases. Given the impact of p16 status, a surrogate marker of the human papillomavirus (HPV) infection, on radiosensitivity, we attempted to apply the RSI and GARD to estimate p16-associated radiosensitivity in head and neck squamous cell carcinoma (HNSC). For this purpose, The Cancer Genome Atlas (TCGA) dataset was employed. In the GARD calculation, we assumed that p16-positive patients received 60 Gy in 30 fractions, while p16-negative patients received 70 Gy in 35 fractions. p16 positivity was associated with favorable characteristics compared to negative patients. The RSI and GARD analyses demonstrated increased radiosensitivity and high therapeutic effect in p16-positive patients, compared to p16-negative patients. Additionally, tumor microenvironmental conditions predicted by other models were also significantly affected by p16 status. Collectively, the models used in this study could be a promising tool for estimating p16-associated radiosensitivity in HNSC.

Stimuli Responsive Molecular Exchange of Structure Directing Agents on Gold Nanobipyramids: Cancer Cell Detection and Synergistic Therapeutics.

Surface-engineered gold nanoparticles have been considered as versatile systems for theranostics applications. Moreover, surface covering or stabilizing agents on gold nanoparticles especially gold nanobipyramids (AuNBPs) provides an extra space for cargo molecules entrapment. However, it is not well studied yet and also the preparation of AuNBPs still remains dependent largely on cetyltrimethylammonium bromide (CTAB), a cytotoxic surfactant. Therefore, the direct use of CTAB stabilized nanoparticles is not recommended for cancer theranostics applications. Herein, we address an approach of dodecyl ethyl dimethylammonium bromide (DMAB) as biocompatible structure directing agent for AuNBPs, which also accommodate anticancer drug doxorubicin (45%), an additional chemotherapeutics agent. Upon near-infrared light (NIR, 808 nm) exposure, engineered AuNBPs exhibit (i) better phototransduction (51 °C) due to NIR absorption ability (650-900 nm), (ii) photo triggered drug release (more than 80%), and (iii) synergistic chemophototherapy for breast cancer cells. Drug release response has been evaluated in tumor microenvironment conditions (84% in acidic pH and 80% at high GSH) due to protonation and high affinity of thiol binding with AuNBPs followed by DMAB replacement. Intracellular glutathione (GSH, 5-7.5 mM) replaces DMAB from AuNBPs, which cause easy aggregation of nanoparticles as corroborated by colorimetric shifts, suggesting their utilization as a molecular sensing probe of early stage cancer biomarkers. Our optimized recipe yield is monodisperse DMAB-AuNBPs with ∼90% purity even at large scales (500 mL volume per batch). DMAB-AuNBPs show better cell viability (more than 90%) across all concentrations (5-500 ug/mL) when directly compared to CTAB-AuNBPs (less than 10%). Our findings show the potential of DMAB-AuNBPs for early stage cancer detection and theranostics applications.

Enhancing Magnetic Hyperthermia Efficacy through Targeted Heat Shock Protein 90 Inhibition: Unveiling Immune-Mediated Therapeutic Synergy in Glioma Treatment.

The induction of heat stress response (HSR) mediated by the generation of heat shock proteins (HSPs) on exposure to magnetic hyperthermia-mediated cancer therapy (MHCT) decreases the efficacy of localized heat treatment at the tumor site, and thus therapy remains a significant challenge. Hence, the present study examined differential HSR elicited in glioma cells post-MHCT under different tumor microenvironment conditions (2D monolayers, 3D monoculture, and coculture spheroids) to recognize target genes that, when downregulated, could enhance the therapeutic effect of MHCT. Gene expression analysis following MHCT revealed that HSP90 was upregulated as compared to HSP70. Hence, to enhance the efficacy of the treatment, a combinatorial strategy using 17-DMAG as an inhibitor of HSP90 following MHCT was investigated. The effects of combinatorial therapy in terms of cell viability, HSP levels by immunofluorescence and gene expression analysis, oxidative stress generation, and alterations in cellular integrity were evaluated, where combinatorial therapy demonstrated an enhanced therapeutic outcome with maximum glioma cell death. Further, in the murine glioma model, a rapid tumor inhibition of 65 and 53% was observed within 8 days at the primary and secondary tumor sites, respectively, in the MCHT + 17-DMAG group, with abscopal effect-mediated complete tumor inhibition at both the tumor sites within 20 days of MHCT. The extracellularly released HSP90 from dying tumor cells further suggested the induction of immune response supported by the upregulation of IFN-γ and calreticulin genes in the MHCT + 17-DMAG group. Overall, our findings indicate that MHCT activates host immune systems and efficiently cooperates with the HSP90 blockade to inhibit the growth of distant metastatic tumors.

Implementing personalized lung cancer care with spatial profiling, patient derived organoids, and rationally designed drug testing.

e20521 Background: Lung cancer (LC) remains the leading cause of cancer-related death in 2024, with most LC diagnoses with non-small cell lung cancer (NSCLC) subtype. Tyrosine kinase inhibitors (TKI) and targeted therapy against oncogenic drivers (KRAS, EGFR, or ALK fusions, others) are effective initial therapies; however, nearly all treated patients eventually develop resistance to therapy. Key reasons are the focus on limited predictive biomarkers and lack of patient derived LC models that reflect the underlying heterogeneity and the various phenotypic cell states among subclones within and between patients. Methods: We report the establishment of a novel platform for personalized LC care by refining the resolution of molecular interrogation of diagnostic and resistance biopsies to the single cell level, and with spatial profiling using single cell spatial multiomic (scSpMO) and coupling these enhanced diagnostic tools with functional validation with rational designed drug screens in patient-derived organoids (PDOs). Results: For deriving LC PDOs in epithelial and tumor microenvironment (TME) conditions to faithfully maintain the histological and genetic features of their respective LC tissues, we developed enrichment conditions for LC PDOs guided by tumor mutation variants and activated pathways. PDOs are maintained under the same treatment condition in the clinic (e.g., Lorlatinib) and drug testing is tailored to identify potentially most effective next lines of therapy. With nine patients enrolled and others in ongoing studies, single cell PDOs (at > 80% establishment rate) were used to determine activated pathways, lineage plasticity and acquired resistance. Functional assays for acquired resistance such as PI3K/AKT signaling, Src kinase, BRAF fusion and MET hyperactivity allow the identification of potentially novel lines of therapy and treatment sequence and/or combinations for each patient, through the compassionate care program and/or trial participation. Conclusions: Our platform when empowered by combining datasets from scRNA-seq and scSpMO signatures, with validated functional drug responses in PDOs, offers the ability to perform high-content assays, predict and/or act on resistance to therapy for each patient, and provides a path for precision medicine-guided impact on patient care.

Tumor microenvironment activated and amplified self-luminescent nano photosensitizers for efficient treatment of triple negative breast cancer

Triple negative cancer (TNBC) is characterized as an aggressive phenotype lacking a specific therapeutic target. To date, self-illuminating photodynamic therapy (PDT) based on chemiluminescence resonance energy transfer (CRET) has emerged as a potential alternative for TNBC treatment by generating singlet oxygen (1O2), overcoming limitations in light penetration. However, this self-illuminating strategy heavily relies on endogenous hydrogen peroxide (H2O2) and oxygen (O2) within the tumor microenvironment (TME), resulting in inefficient therapeutic performance. In this study, we designed CLT@DPD nanoparticles capable of self-illumination via CRET and TME regulation through self-supplying H2O2 and O2. This nanoparticle was constructed by encapsulating luminol-tetreaphenylporphyrin conjugate (LT) and nanoscale CaO2 with DSPE-PEG 2000 and dioleoylphosphatidylcholine (DOPC). A good accumulation at tumor site was achieved due to enhanced permeability and retention (EPR) effect after intravenous injection of CLT@DP. Responsive to the high H2O2 levels and acidic aqueous conditions of TME, LT can be oxidized to produce 1O2via CRET, and the CaO2 can be decomposed to supply H2O2 and O2. Additionally, the presence of DOPC helps to enhance the permeability of the micelle shell under the oxidation of 1O2, thereby accelerating the release of H2O2 and O2. Our proposed nanoparticles exhibit excellent performance in eradicating in situ tumor cells and inhibiting metastasis by simultaneously enhancing self-luminous PDT and alleviating oxygen depletion in TME.

Nano-assemblies overcome cancer multidrug resistance for effectively synergistic chemo-immuno-oncotherapy

The combination of chemotherapy and immunotherapy is the first line treatment for many cancers. However, resistance to chemotherapy is a common phenomenon, as well as the failure of antigen presentation and T cell infiltration are the fundamental obstacle for effective combined oncotherapy. Many studies have clarified the importance of increased reactive oxygen species (ROS) in reducing multidrug resistance (MDR) by disrupting oxidative balance and activating signaling pathways. Here, to modulate immunosuppressive tumor microenvironment (TME) and overcome the cancerous resistance to chemotherapy for effectively tumor killing, the SIM@NPs-PDA/DOX nano-assemblies, was developed to sequentially delivery doxorubicin (DOX), polydopamine (PDA) and simvastatin (SIM) for tumor chemo-immunotherapy. When the nano-assemblies penetrated into tumor TME, PDA and DOX were released by the hypoxia and low acid condition of TME, which sensitized the tumor cells for reducing the tumor resistance against DOX and effectively induced immunogenic tumor cell death (ICD), respectively. The neo-antigen generated by the ICD effect in situ could be greatly presented by the SIM activated dendritic cells (DCs) for specific T cells production. Simultaneously, the released DOX was also expected to kill tumor-associated macrophages (TAMs) for enhancing the specific T cell infiltration. In B16F10 and MCF-7 tumor cells with acquired drug resistance and relevant tumor models, the designed nano-assemblies showed powerful anti-tumor effects by reducing MDR, killing cancer cells and TAMs, promoting CD8+ T cells activation and infiltration. This study provides a novel nano-assemblies for effective onco-immuno-chemotherapy by overcoming cancer multidrug resistance and immuno-depletion.

Multienzyme-Like Polyoxometalate-Based Single-Atom Enzymes for Cancer-Specific Therapy Through Acid-Triggered Nontoxicity-to-Toxicity Transition.

Single-atom enzymes (SAzymes) exhibit great potential for chemodynamic therapy (CDT); while, general application is still challenged by their instability and unavoidable side effects during delivery. Herein, a manganese-based polyoxometalate single-atom enzyme (Mn-POM SAE) is first introduced into tumor-specific CDT, which exhibits tumor microenvironment (TME)-activated transition of nontoxicity-to-toxicity. Different from traditional POM materials, the aggregates of low-toxic Mn-POM SAE nanospheres are obtained at neutral conditions, facilitating efficient delivery and avoiding toxicity problems in normal tissues. Under acid TME conditions, these nanospheres are degraded into smaller units of toxic Mn(II)-PW11; thus, initiating cancer cell-specific therapy. The released active units of Mn(II)-PW11 exhibit excellent multienzyme-like activities (including peroxidase (POD)-like, oxidase (OXD)-like, catalase (CAT)-like, and glutathione peroxidase (Gpx)-like activities) for the synergistic cancer therapy due to the stabilized high valence Mn species (MnIII/MnIV). As demonstrated by both intracellular evaluations and in vivo experiments, ROS is generated to cause damage to lysosome membranes, further facilitating acidification and impaired autophagy to enhance cancer therapy. This study provides a detailed investigation on the acid-triggered releasing of active units and the electron transfer in multienzyme-mimic-like therapy, further enlarging the application of POMs from catalytical engineering into cancer therapy.

Abstract 2906: Inducing and disabling COX-2 S-nitrosylation to investigate its role in breast cancer progression

Abstract Background: We aim to assess the effect of S-nitrosylation of COX-2 on tumor attributes in models of early breast cancer. COX-2 is involved in tumor progression in many solid cancers. It is a known oncogene in murine breast cancer models, and targetable by NSAIDs. In women, its clinical correlation to breast cancer progression as well as its efficacy as a therapeutic target have yielded mixed results. These incongruities may be due, in part, to a post-translational modification, as COX-2 undergoes S-nitrosylation on Cys-526. Commercially-available antibodies selectively bind to either the S-nitrosylated (SNO-COX-2) or non-nitrosylated (nCOX-2) forms, and immunohistochemical staining of human tissue reveals that the two forms of COX-2 localize to different cellular sites in normal breast tissue and have opposite associations with breast cancer progression (PMID 33298921). Hypothesis: S-Nitrosylated and non-nitrosylated COX-2 have distinct roles in cancer progression. Approach: MCF10DCIS.com is a human breast cancer cell line chosen due to its ability to form DCIS-like (“stage 0”) lesions in culture and xenografting, which can progress to invasion when induced by signals from the tumor microenvironment (TME). Approach 1: Detect extracellular matrix (ECM) and ligand factors that upregulate SNO-COX-2, independently or in ECM/ligand pairs. We utilized a 2D microenvironment microarray (MEMA) and tested 357 TME conditions. Approach 2: Validate TME conditions in 3D culture. Approach 3: Develop a mutant MCF10DCIS.com line in which COX-2 cannot be S-nitrosylated by inducing a single disabling missense mutation at Cys-526 via CRISPR. Results: The MEMA revealed several conditions in which SNO-COX-2 was upregulated, including fibular collagens and SSP1. In 3D culture, increased SNO-COX-2 was associated with the presence of markers for EMT and an invasive growth phenotype, whereas nCOX-2 was not. Following CRISPR transformation, 59 individual MCF10DCIS.com clones were isolated and the target site of each sequenced. We identified 10 heterozygotic clones with the desired mutation, including a classical heterozygote that also retained the WT allele. Ongoing: Current studies include a second round of CRISPR to produce a homozygous mutant cell line as well as validation of the mutant protein. The resulting cell lines will model the loss of COX-2 S-nitrosylation in breast cancer cells while retaining otherwise-functional COX-2. Citation Format: Reuben J. Hoffmann, AeSoon Bensen, Elise de Wilde, James E. Korkola, Pepper Schedin. Inducing and disabling COX-2 S-nitrosylation to investigate its role in breast cancer progression [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 2906.

Abstract 6496: Assessment of tyrosine kinase inhibitor bypass mechanisms in lung cancer using single cell multiomics, patient derived organoids and rationally designed drug screens

Abstract Lung cancer (LC) is the leading cause of cancer-induced mortality, with 350 patients in the US dying from LC daily, the majority from non-small cell lung cancer (NSCLC). Molecular-targeted therapy such as tyrosine kinase inhibitors (TKI) are effective in first and subsequent lines of therapy against oncogenic drivers (KRAS, EGFR, or ALK fusions, others); however, nearly all treated patients progress eventually and run out of therapeutic options. Key reasons are the lack of predictive biomarkers and LC models that reflect the underlying heterogeneity and different phenotypic cell states among subclones within and between patients. Here, we developed a novel platform for refining the resolution of molecular interrogation to the single cell level, using single cell spatial multiomic (scSpMO), coupled with rational designed drug screens in patient-derived organoids (PDOs) from LC biopsies obtained upon progression. By deriving LC PDOs in epithelial and tumor microenvironment (TME) conditions to faithfully maintain the histological and genetic features of their respective LC tissues, we identified enrichment conditions for LC PDOs guided by tumor mutation variants and activated pathways. PDOs are maintained under the same treatment condition in the clinic (e.g., Lorlatinib) and drug testing is tailored to identify next lines of combined therapy. With six patients enrolled and others in ongoing studies, single cell PDOs (at >80% establishment rate) were used to determine key molecular bypass mechanism for resistance to TKI, including on-target variants, driver bypass, lineage plasticity and acquired resistance. Functional assays for acquired resistance such as PI3K/AKT signaling, Src kinase, BRAF fusion and MET hyperactivity allow the identification of potential novel combined lines of therapy for each patient through the compassionate care program and/or trial participation. Our platform when empowered with datasets of bulk, scRNA-seq and scSpMO signatures of primary human NSCLC, together with PDOs offer the ability to perform analysis of LC phenotypes such as lineage transformation in high-content assays, predict and/or act on resistance to therapy and provides a path for precision medicine-guided immediate impact on patient care. Citation Format: Liqiong Liu, Ann Strange, Schuyler Lee, Bifeng Gao, Daniel Merrick, Tejas Patil, James DeGregori, D Ross Camidge, Sharon R. Pine, Hatim E. Sabaawy. Assessment of tyrosine kinase inhibitor bypass mechanisms in lung cancer using single cell multiomics, patient derived organoids and rationally designed drug screens [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 6496.

Abstract 4240: A comprehensive in vitro tumor model: One-stop simultaneous isolation and culture of tumoroids, CAFs and TILs for advancing immuno-oncology research

Abstract The tumor microenvironment (TME), composed of a diverse array of cells, jointly participates in tumor formation and has attracted attention as a direct or indirect therapeutic target. Among the tumor-forming cells, cancer cells, cancer-associated fibroblast (CAF), and tumor-infiltrating lymphocytes (TILs) comprise major proportion and players in tumor biology. Therefore, an in vitro tumor model composed of at least above three major cellular players better mimics a patient’s tumor. Here, we developed a patient-derived tumor model containing tumoroids, CAFs and TILs isolated from the patient’s tumor tissue by using one-stop isolation culture system. Our one-stop culture system utilizes a Transwell-based method that concurrently segregates cancer cells, CAFs, and TILs based on distinct characteristics such as cell size, migration speed, and growth. Lung tumoroids, fibroblasts, and TILs segregated in Matrigel-coated upper Transwell chamber, Transwell pores and in the lower chamber, respectively. Lung tumoroids maintain the patient’s lung cancer tissue characteristics and genetic features during extended in vitro cultivation. Fibroblasts extracted from the Transwell pores exhibit significant similarity to CAFs found in patient tissue. TILs in the lower part of the Transwell chamber closely resemble those found in the original cancer tissue analyzed by FACS. We have also refined the functionality of TILs through a two-week expansion process. Patients’ tumor composed of 0.15 to 95.3 % of EpCAM positive tumor cells, 0.14 to 19.4 % of CD90 positive fibroblasts and 3.98 to 91.2 % of immune cells in FACS analysis. By reassembling three main cellular components of the tumor in a proper proportion, we developed a co-culture platform for a patient-derived tumor model reproducing patient-specific TME conditions. The co-culture platform showed a potential not only for an experimental model but also to use a predictive test of personalized immunotherapy responses for lung cancer patients. Citation Format: YOO RI KO, Sung Min Kim, Hye Seon Park, Se Jin Jang. A comprehensive in vitro tumor model: One-stop simultaneous isolation and culture of tumoroids, CAFs and TILs for advancing immuno-oncology research [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 4240.

Nanoparticle-Catalyzed Transamination under Tumor Microenvironment Conditions: A Novel Tool to Disrupt the Pool of Amino Acids and GSSG in Cancer Cells.

Catalytic cancer therapy targets cancer cells by exploiting the specific characteristics of the tumor microenvironment (TME). TME-based catalytic strategies rely on the use of molecules already present in the TME. Amino groups seem to be a suitable target, given the abundance of proteins and peptides in biological environments. Here we show that catalytic CuFe2O4 nanoparticles are able to foster transaminations with different amino acids and pyruvate, another key molecule present in the TME. We observed a significant in cellulo decrease in glutamine and alanine levels up to 48 h after treatment. In addition, we found that di- and tripeptides also undergo catalytic transamination, thereby extending the range of the effects to other molecules such as glutathione disulfide (GSSG). Mechanistic calculations for GSSG transamination revealed the formation of an imine between the oxo group of pyruvate and the free -NH2 group of GSSG. Our results highlight transamination as alternative to the existing toolbox of catalytic therapies.

Sequential dual-locking strategy using photoactivated Pt(IV)-based metallo-nano prodrug for enhanced chemotherapy and photodynamic efficacy by triggering ferroptosis and macrophage polarization

Selective activation of Pt(IV) prodrugs within tumors has emerged as a promising strategy in tumor treatment. Although progress has been made with photo- and ultrasound-activated Pt(IV) prodrugs, concerns remain over the non-specific activation of photosensitizers (PS) and the potential for phototoxicity and chemical toxicity. In this study, a sequential dual-locked Pt(IV) nano-prodrug that can be activated by both the acidic tumor microenvironment and light was developed. The Pt(IV) prodrug was prepared by conjugating PS-locked Pt(IV) to a polymeric core, which was then chelated with metallo iron to lock its photoactivity and form a metallo-nano prodrug. Under acidic tumor microenvironment conditions, the metallo-nano prodrug undergoes dissociation of iron, triggering a reduction process in oxaliplatin under light irradiation, resulting in the activation of both chemotherapy and photodynamic therapy (PDT). Additionally, the prodrug could induce metallo-triggered ferroptosis and polarization of tumor-associated macrophages (TAM), thereby enhancing tumor inhibition. The dual-lock strategy employed in a nanoparticle delivery system represents an expansion in the application of platinum-based anticancer drugs, making it a promising new direction in cancer treatment.