Tumor Model Research Articles

Development of Mg‐Alginate Based Self Disassociative Bio‐Ink for Magnetic Bio‐Patterning of 3D Tumor Models

AbstractAlginate forms a hydrogel via physical cross‐linking with divalent cations. In literature, Ca2+ is mostly utilized due to strong interactions but additional procedures are required to disassociate Ca‐alginate hydrogels. On the other hand, Mg‐alginate hydrogels disassociate spontaneously, which might benefit certain applications. This study introduces Mg‐alginate as the main component of a bio‐ink for the first time to obtain 3D tumor models by magnetic bio‐patterning technique. The bio‐ink contains magnetic nanoparticles (MNPs) for magnetic manipulation, Mg‐alginate hydrogel as a sacrificial material, and cells. The applicability of the methodology is tested for the formation of 3D tumor models using HeLa, SaOS‐2, and SH‐SY5Y cells. Long‐term cultures are examined by Live/dead and MTT analysis and revealed high cell viability. Subsequently, Collagen and F‐actin expressions are observed successfully in 3D tumor models. Finally, the anti‐cancer drug Doxorubicin (DOX) effect is investigated on 3D tumor models, and IC50 values is calculated to assess the drug response. As a result, significantly higher drug resistance is observed for bio‐patterned 3D tumor models up to tenfold compared to 2D control. Overall, Mg‐alginate hydrogel is successfully used to form bio‐patterned 3D tumor models, and the applicability of the model is shown effectively, especially as a drug screening platform.

Abstract IA019: Regulation of EMT tumor states by stromal cells

Abstract Epithelial-to-mesenchymal transition (EMT) is a reversible process during which epithelial cells lose their epithelial characteristics and acquire mesenchymal features, that are important for tumor initiation, progression, stemness, metastasis, and therapy resistance. Different EMT states, each with distinct functional properties, have been identified across various tumor models. These EMT states reside within specific physical niches, characterized by unique stromal cell populations. Understanding the cellular interactions within the tumor microenvironment (TME) and their influence on EMT could lead to novel therapeutic strategies. To understand how the TME regulates the distinct tumor states, we performed single-cell RNA sequencing of the tumor cells and their TME in mouse cancer models presenting EMT. We identified distinct subsets of cancer-associated fibroblasts (CAFs), macrophages, neutrophils, endothelial cells, pericytes, adipocytes, dendritic cells, mast cells, Schwann cells, and T cells in different tumor types. To unravel how TME-tumor cell interactions regulates EMT, we used different bioinformatic approaches to identify ligand-receptor pairs that are likely to organize the tumor niche and to regulate tumor transition states. In vitro treatment of tumor cells with some of these ligands demonstrated their EMT-promoting effects. These findings provide novel mechanistic insights on the crosstalk between tumor cells and the surrounding TME, that may have important implications for anti-cancer therapy. Citation Format: Cédric Blanpain. Regulation of EMT tumor states by stromal cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr IA019.

Abstract B014: NRF2 regulation of the immune microenvironment in breast cancer

Abstract Immune checkpoint inhibitors (ICIs) have emerged as a significant advance in the treatment of various cancer types, including breast cancer, though their efficacy varies widely depending on the cancer subtype and the stage of the disease. Following their initial approval in metastatic triple-negative breast cancer (TNBC), ICIs have shown promising results in other contexts, including early-stage cancers and ER+ and HER2+ subtypes. However, even in advanced TNBC the objective response rate to monotherapy ICI is only ∼20%. These results highlight the need to better understand determinants of ICI response in breast cancer. A detailed, high-resolution definition of the tumor immune microenvironment (TIME) will be critical for predicting and engineering effective responses to ICIs. In previously published work, we and others found that the antioxidant transcription factor NRF2 is constitutively active in breast cancer and is associated with tumor recurrence, metastasis, and poor prognosis. However, the mechanisms through which NRF2 promotes breast cancer progression remain unclear. In the current study, we explore the role of NRF2 in reshaping the TIME and its impact on breast cancer progression using preclinical models, flow cytometry, and transcriptomic analyses. We find that NRF2 is constitutively activated in a subset of human and mouse breast cancers where it functions to suppress inflammatory gene expression. In models of recurrent and metastatic mammary tumors, NRF2 knockdown results in a marked reduction in tumor growth rates. Furthermore, the NRF2 inhibition significantly alters the composition of innate and adaptive immune cell populations within the tumor microenvironment. Cytokine profiling revealed that these immune alterations are associated with increased levels of inflammatory cytokines in NRF2-deficient tumor cells compared to controls. RNA sequencing of tumor cells further supports the observed shifts in the immune landscape associated with NRF2 deficiency. Collectively, our findings demonstrate that the loss of NRF2 impairs tumor growth in part through remodeling the TIME and suggest that NRF2 may be a promising therapeutic target for sensitizing breast cancers to ICIs. Citation Format: Yasemin Ceyhan Ozdemir, James V. Alvarez. NRF2 regulation of the immune microenvironment in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B014.

Abstract C020: Discovery & characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors

Abstract While adoptive cellular therapies (ACT) such as CAR-T and NK therapy have shown clinical efficacy against hematologic malignancies, their use against solid tumors (such as ovarian or pancreatic cancer) has had limited success to date. In particular, CAR-T therapy for solid cancers has led to severe toxicities, including on-target, off-tumor killing of healthy cells and graft vs. host disease (GVHD) with allogeneic transplant. NK cell therapies, in contrast, have fewer associated toxicities due to specific and innate recognition/killing of cancer cells. This greater safety profile makes NK therapies an attractive treatment option for solid cancers. NK therapies are currently limited by insufficient in vivo tumor clearance, caused largely by their lower cytotoxicity and in vivo expansion compared to CAR-T cells. To address this, we have screened 300,000 small molecule compounds for their ability to enhance NK cytotoxicity against ovarian cancer (OVCAR) cells. Our primary screen identified 11 lead compounds which increase NK killing activity against OVCAR cells >= 10% over baseline (vehicle-treated control). Secondary in vitro assays have confirmed that at least 3 out of 11 lead compounds enhance NK cytotoxicity by at least 10% over baseline. As such, we hypothesize that our lead compounds will increase in vivo NK cytotoxicity and tumor clearance, through mechanisms which might include I) upregulation of NK activating receptors, II) downregulation of NK inhibitory receptors, and/or III) modulation of NK metabolism in the immunosuppressive tumor microenvironment. Immediate future studies include in vivo testing of our lead compounds’ ability to enhance NK cytotoxicity, using immunodeficient mouse models of subcutaneous OVCAR tumors. Moreover, we will utilize 2D and 3D in vitro culture techniques alongside flow cytometry and RNA sequencing to identify how lead compounds impact markers of NK activation and metabolism. This will include expression of activating/inhibitory receptors, exhaustion markers, and key metabolic factors. Finally, we will assess the effect of lead compounds on NK function within an immunosuppressive tumor environment. Our findings will contribute to effective future NK cell therapies for solid tumors. Citation Format: Indrani Das, Olubukola Abiona, Riufu Liu, Grace K. Lee, David N. Wald. Discovery & characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C020.

Abstract B038: Establishing a screening platform to identify key signaling molecules regulating cold tumor formation through immune evasion and exclusion

Abstract Many trial data have shown that cancer immunotherapy can successfully induce long lasting anti-cancer immune responses and increased the survival rate of cancer patients. One of the key challenges facing cancer immunotherapy is the low response rate of cancer patients to immunotherapy, suggesting that these cancer patients may have acquired resistance toward immunotherapy before treatments. Therefore, there is an unmet need for identifying the underlying mechanisms that promote resistance. One important hypothesis is that immune-excluded tumors (so called cold tumors) that lack of functional T cells have been often found in cancer patients who cannot respond well to cancer immunotherapy. To address this, we have successfully established a preclinical model for cold lung tumors, conducted RNA-seq, performed hallmark gene set enrichment analysis (GSEA) on cold tumor cells sorted in vivo, and identified several candidates associated with epithelial-mesenchymal transition, Wnt signaling, type I and II interferon (IFN) signaling, and p53 signaling. We hypothesize that their roles may either promote or inhibit the formation of cold tumors. One of the molecular candidates we identified, a molecule designated as IRC001, represents a crucial proof of concept for our experimental tumor model. We have unveiled a novel role for IRC001 in enhancing anti-tumor immunity, leading to the transformation of cold tumors into hot tumors and facilitating swift tumor regression through pathways reliant on T cells and type I IFN signaling. Our mechanistic studies have shown that IRC001 can trigger type I IFN signaling by engaging STING/TBK-1/IRF3-dependent pathways. To delve deeper into the spatial and molecular distinctions in mouse tumors (± IRC001), we initiated a preliminary study utilizing 10x Genomics/Visium to examine the spatial variations between these tumor types. Significantly, our spatial transcriptomic analysis revealed consistent immune-excluded and immune-enriched characteristics compared to traditional RNA sequencing methods. We conducted an analysis of TCGA datasets (The Cancer Genome Atlas) and observed that elevated IRC001 expression is associated with improved prognosis in SKCM (Skin Cutaneous Melanoma). Furthermore, our bioinformatics analysis revealed that melanoma patients exhibiting heightened IRC001 expression displayed positive responses to immune checkpoint blockade therapies. Therefore, we believe that our experimental tumor model can provide vital insights into the cold tumor microenvironment, shaping future therapeutic strategies, and pinpointing molecular candidates that can assist in selecting patients for successful immunotherapy. Citation Format: Yi-Ting Liao, Li-Mei Chen, Wen-Jye Lin. Establishing a screening platform to identify key signaling molecules regulating cold tumor formation through immune evasion and exclusion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B038.

Abstract B036: WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway

Abstract Immune checkpoint blockade (ICB) has revolutionized cancer treatment, but resistance limits its clinical effectiveness. Therefore, understanding immune resistance mechanisms and identifying predictive markers are essential. Our transcriptomic analysis of PD-1 blockade-treated patients and non-responsive tumor models identifies WEE1 as a key resistance factor, promoting cancer stem cell (CSC)-like traits and immune resistance phenotypes including low T cell infiltrated into tumor and anti-apoptotic properties to cytotoxic T cells (CTLs). WEE1 drives AKT hyperactivation, enhancing expression of resistance factors such as CYCLIN A which contributes to CSC-like properties, and the anti-apoptotic molecule MCL-1, while also reducing T cell infiltration via CXCL10 downregulation. This signaling axis is conserved across various cancers. Notably, WEE1 inhibition sensitizes tumors to ICB by disrupting these refractory properties and reinvigorating antitumor immunity. These findings underscore the novel role of WEE1 in driving immune resistance and CSC-like characteristics, supporting the use of WEE1 inhibitors as a strategy to overcome resistance to anti-PD-1 therapy. Citation Format: Hyo-Jung Lee, Suyeon Kim, Eunho Cho, Tae Woo Kim. WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B036.

Abstract C047: AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial

Abstract Introduction: TM4SF1 (Transmembrane-4 L-Six-Family-Member-1) is an endothelial marker with critical roles in angiogenesis, as well as a tumor cell antigen that contributes significantly to invasion and metastasis. TM4SF1 is upregulated 20-fold in angiogenic tumor vascular endothelium compared to normal vasculature endothelium and exhibits a unique nuclear internalization pathway. AGX101 is a novel tubulin inhibitor conjugate specifically directed against TM4SF1, delivering a potent maytansinoid payload directly to the nucleus of cells within the tumor microenvironment. Delivery to both the vascular and tumor cell compartments of the tumor results in three mechanisms of action (MoAs): (1) activation of tumor immune surveillance, (2) tumor blood supply deprivation, and (3) direct tumor cell killing. Methods: TM4SF1 expression was assessed across solid tumor samples using immunohistochemistry. Safety and pharmacokinetics of AGX101 were evaluated in non-human primates, with escalating doses to determine the highest non-severely toxic dose (HNSTD). Efficacy studies were also conducted in mouse models, enabling assessment of the minimum effective dose (MED) needed to engage each of the three MoAs. The combination of HNSTD and MED enables calculation of a therapeutic index (TI). Preclinical efficacy studies used either AGX101 (in human tumor xenograft models, to study the tumor cell killing MoA) or AGXB01, a rodent surrogate of AGX101, to study vascular and immune MoAs and all three MoAs in syngeneic mouse cancer models. The potential for synergy with immune checkpoint inhibitors (ICIs) was also investigated. A first-in-human study of AGX101 in patients with advanced solid tumors with the primary objective of safety and dose-limiting toxicities (DLTs) has initiated. Results: TMFS41 is broadly expressed across solid tumors with increasing scoring intensity in higher grade tumors. In non-human primates, AGX101 exhibited a favorable safety profile, being tolerated at relatively high doses and with dose dependent, monitorable, and reversible effects. In preclinical efficacy studies, AGXB01 and AGX101 as monotherapies demonstrated robust efficacy through each of the three MoAs in multiple syngeneic, xenograft, and orthotopic tumor models in mice and rats as well as non-tumor models of angiogenesis. Measured by exposure, TI was large. Additionally, AGXB01 was shown to potentiate the effects of ICIs in syngeneic mouse models (CT26, Renca, and B16-F10), suggesting a potential synergistic approach in cancer therapy. The first dose level of AGX101 monotherapy in humans has cleared without DLTs. Conclusion: AGX101, targeting the TM4SF1 antigen, represents a promising new approach in cancer therapy. The preclinical data suggest that AGX101 could provide a significant therapeutic benefit by novel and differentiated mechanisms of action, namely selectively targeting the tumor vasculature and potentiating immune-based therapies. Further clinical development of AGX101 is ongoing. Citation Format: Ildefonso Ismael Rodriguez Rivera, Meredith S. Pelster, Shou-Ching Jaminet, Paul Jaminet, Glen J. Weiss. AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C047.

Abstract C007: Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most aggressive type of pancreatic cancer, is predicted to become the second leading cause of cancer related deaths by 2030. Interleukin 17 (IL-17) signaling can promote tumor growth. In pancreatic cancer, IL-17 signaling in cancer cells is capable of triggering the formation of neutrophil extracellular traps (NETs) which is detrimental to the anti-tumor effect of immunotherapy. IL-17 signaling has been shown to have critical roles in maintaining gut homeostasis. Intestinal epithelial cells directly interact with the gut microbiome and are actively involved in infection control, injury repair, immune responses, and the production of antimicrobial peptides and cytokines, and need to be constantly replenished by intestinal stem cells (ISCs). Our lab’s study has previously shown that deletion of Interleukin 17 receptor A (IL-17RA) in the entire gut epithelium induces pro-tumorigenic IL-17 signaling due to the disruption of gut homeostasis, suggesting that immune responses induced by gut IL-17/IL-17RA signaling can affect the behavior of distant tumors. However, the mechanism of IL-17 signaling within specific gut epithelial compartments such as ISCs, enterocytes, secretory cells, etc. and their role in maintaining gut homeostasis and cancer risk are not well-studied until now. Thus, we aimed to identify the importance of IL-17 signaling in ISCs during pancreatic cancer. We hypothesized that disruption of IL-17/IL-17RA signaling within ISCs impacts tumor growth. Our preliminary data shows that IL-17 signaling in enterocytes maintains intestinal homeostasis and disruption of this axis triggers systemic increase in B cells that promotes tumor growth by increasing IL-17 levels in circulation. To identify the function of IL-17/IL-17RA signaling in ISCs, we utilized Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), a lineage marker for ISCs, to drive Cre enzyme tissue specific expression to specifically knockout Il17ra in gut ISCs - an inducible transgenic mouse model Il17ra fl/fl ;Lgr5- EGFP-CreERT2 (cKO). We used multiplex staining and imaging to validate the cell type specific loss of IL-17RA expression in tamoxifen treated cKO mice. We used implantable pancreatic tumor models and performed immunoprofiling using flow cytometry. We found that the pancreatic tumor growth was altered by the remote signal from gut ISCs in cKO mice. Furthermore, we performed RNAseq in both tumor and gut tissue which identified that the major changes in gene expression were associated with both innate and adaptive immune pathways, especially B cell activation. We conclude that the IL-17/IL-17RA signaling in gut ISCs changes intestinal immune responses locally that can affect distant tumor immunity. Citation Format: Haoyue Liu, Vidhi Chandra, Le Li, Olivereen Le Roux, Virginia Tahan, Florencia McAllister. Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C007.

Abstract C005: Tumor vaccines and oncolytic virotherapy: systemic immunity vs. tumor microenvironment

Abstract Tumor vaccines, particularly the innovative mRNA variants, hold immense promise in cancer immunotherapy. Despite their potential, these vaccines encounter formidable challenges in surmounting immune tolerance, the immune-suppressive tumor microenvironment (TME) and more importantly, the resistance developed in cancer cells that are antigen negative. Concurrently, oncolytic virotherapy (OV), a distinct facet of tumor vaccination, significantly alters the TME and releases tumor antigens via lysis to generate a broader spectrum of tumor antigens. This unique property positions OV to induce anti-tumor immunity, as evidenced by prolonged overall survival and abscopal tumor suppression. Recognizing the complementary strengths of mRNA tumor vaccines and OV, we propose a combined approach to address the limitations inherent in each modality. Our study tested this hypothesis by combining a mRNA vaccine targeting HER2 with oncolytic viruses, utilizing a prime-boost strategy on a HER2-expressing CT26 mouse tumor model. Notably, while the mRNA vaccine alone induced specific anti-HER2 systemic immunity, the combination demonstrated a potent immune response against both anti-HER2 and anti-CT26, leading to durable tumor eradication. Our findings highlight that peripheral vaccination has the potential to elicit an immune response in non-immune suppressive environments. However, combining it with virotherapy not only amplifies specific immunity but also induces a broader immune response against a spectrum of tumor antigens. This synergistic combination represents a promising avenue for advancing cancer immunotherapy, offering the potential for improved treatment outcomes and broader applicability of tumor vaccines across diverse cancer types. Citation Format: William Jia. Tumor vaccines and oncolytic virotherapy: systemic immunity vs. tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C005.

PLLA Porous Scaffold as a 3D Breast Cancer Model to Investigate Drug Resistance.

Multidrug resistance remains one of the major challenges in breast cancer research, often leading to treatment failure. To better understand this mechanism, sophisticated three-dimensional (3D) tumor models are necessary, as they offer several advantages over traditional bidimensional (2D) cultures. In this study, poly-l-lactic-acid porous scaffolds were produced using a thermally induced phase separation technique and employed as 3D models for breast cancer cell lines: MDA-MB-231, MCF-7, and its multidrug-resistant variant, MCF-7R. The MTS assay was used to compare growth inhibition following doxorubicin treatment in 2D and 3D. Remarkably, the IC50 values increased in 3D cultures compared to 2D: MDA-MB-231 (445 vs. 54.5 ng/mL), MCF-7 (7.45 vs. 0.75 μg/mL), and MCF-7R (165 vs. 39 μg/mL). MCF-7R, which usually shows greater resistance in 2D, demonstrated even higher resistance in 3D. In fact, IC50 was not reached within 3 days as with the other models, but only after 6 days. Cellular morphology also played a crucial role. When treated with concentrations higher than the IC50, MDA-MB-231 cells lost their physiological 3D clustered structure, while MCF-7 and its resistant variant exhibited disrupted layers. All cell lines in 3D showed higher chemoresistance, suggesting a more biomimetic spatial architecture. Our work bridges the gap between monolayer and animal models, highlighting the potential of polymeric 3D scaffolds in breast cancer research.

Radioiodinated Nanobody immunoPET probe for in vivo detection of CD147 in pan-cancer.

To develop the extracellular matrix metalloproteinase inducer (CD147)-targeting therapeutic strategies, accurate detection of CD147 expression in tumors is crucial. Owing to their relatively low molecular weights and high affinities, nanobodies (Nbs) may be powerful candidates for cancer diagnosis and therapy. In this study, we developed a novel CD147-targeted nanobody radiotracer, [124I]I-NB147, which provides guidance for the noninvasive detection of CD147-overexpressing cancers. CD147 expression in human cancers was detected via immunohistochemistry (IHC) on tissue microarrays (TMAs). Western blot (WB) and flow cytometry were used to screen CD147-positive malignant melanoma (MM), triple-negative breast cancer (TNBC), and pancreatic cancer (PCA) cell lines. The CD147 nanobody (NB147) was labeled with [124I]INa using Iodogen as the oxidizing agent and was purified by the PD-10 column. The physicochemical properties, affinity, metabolic characteristics, biodistribution, and immunoPET imaging of [124I]I-NB147 were evaluated Moreover, [18F]F-FDG was used as a control. Finally, CD147 expression analysis was performed via multiplex immunofluorescence (MxIF) and autoradiography on human cancer specimens and tumor-bearing mice tissues. TMAs results revealed that CD147 is highly expressed in MM, TNBC, and PCA. A CD147-specific nanobody, NB147, was successfully generated with excellent in vitro binding characteristics. [124I]I-NB147 was obtained with high radiochemical yield and purity, and was stable for at least 4h in vitro. WB and FCM revealed that CD147 was positive in A375, MDA-MB-435 and ASPC1 cells, whereas SK-MEL-28, 4T1 and BXPC3 cells presented low expression levels. The radio-ELISA results indicated that [124I]I-NB147 had a high binding affinity to CD147. The uptake of [124I]I-NB147 was significantly different between CD147 high-expression cells and CD147 low-expression cells (P < 0.001). The biological half-life of the distribution and clearance phases were 0.05h and 1.58h, respectively. In CD147-positive tumor models, the [124I]I-NB147 accumulated in A375, MDA-MB-435, and ASPC1 tumors, and the uptake value was significantly higher than that of [18F]F-FDG. Uptake in SK-MEL-28, BXPC3, and 4T1 tumors was not clearly observed. Finally, through autoradiography and histological studies, the correlation analysis between tumor uptake and CD147 expression level was determined. The high expression of CD147 in MM, TNBC, and PCA tissuesand in tumor cells was verified. The CD147 nanobody, NB147 was produced and radiolabeled to obtain the immunoPET probe, [124I]I-NB147, which showed high affinity to CD147 and precise visualization for accurate diagnosis of CD147-expressing lesions in different cancers. These results provide insight into the imaging and binding properties of nanobody NB147 over extended periods of time, reinforcing its potential in developing radionuclide therapies for CD147-positive cancer patients.

Study on the mechanism of hyperoside in affecting the biological progression and radiosensitivity of esophageal carcinoma by modulating the STAT3/AKT/ERK pathway

Hyperoside (HYP) exhibits diverse pharmacological effects and holds potential for enhancing chemotherapy sensitivity. However, few studies have reported the impact of HYP on the malignant progression of esophageal carcinoma (EC) and its sensitivity to radiotherapy. The impact of HYP on the viability of esophageal carcinoma cells (TE-1 and KYSE-150) was assessed using Cell Counting Kit-8 (CCK-8) assays. The biological characteristics and radiosensitivity of esophageal carcinoma cells following HYP treatment were evaluated through colony formation experiments, flow cytometry, scratch wound-healing assays, and transwell migration and invasion assays. Western blot analysis was performed to determine the levels of proteins associated with cell death and epithelial-mesenchymal transition (EMT), as well as to explore whether HYP interferes with the radiosensitivity of esophageal carcinoma cells via the STAT3/AKT/ERK pathways. Finally, a subcutaneous graft tumor model was constructed to investigate the effects of HYP and X-ray treatments on in vivo tumor growth. The findings indicated a dose-dependent decrease in the survival rate of KYSE-150 and TE-1 cells following HYP treatment. HYP treatment also inhibited cell proliferation, invasion, migration, and EMT, while increasing the apoptotic rate and radiosensitivity of the cells. Notably, HYP suppressed the malignant progression of EC and enhanced radiosensitivity via the STAT3/AKT/ERK pathway. Moreover, HYP impaired the growth of esophageal carcinoma tumors in mice, with the combined HYP and X-ray treatment exerting a stronger inhibitory effect. In conclusion, HYP increases the radiosensitivity of esophageal carcinoma cells, offering considerable promise for application in the clinical treatment of EC.

EYA3 promotes the tumorigenesis of gastric cancer through activation of the mTORC1 signaling pathway and inhibition of autophagy.

Gastric cancer (GC) is a leading cause of cancer-related mortality, with a high rate of postoperative recurrence and poor long-term survival. The Eyes Absent (EYA) protein family plays a significant role in cancer progression, with EYA3 being implicated in promoting GC cell proliferation and tumor growth. Utilizing the DepMap database, we identified EYA3 as a gene of interest in GC. We analyzed EYA3 expression in GC tissues and cell lines, performed in vitro assays to assess its role in cell proliferation, and conducted gene set enrichment analysis to explore its relationship with autophagy and the mTORC1 signaling pathway. In vivo, we used a xenograft tumor model to examine the effects of EYA3 expression on tumor progression. EYA3 was consistently upregulated in GC tissues, and its high expression correlated with a decrease in patient survival rates. Silencing EYA3 in GC cell lines resulted in reduced cell proliferation. Inhibition of autophagy and activation of the mTORC1 signaling pathway were observed as mechanisms by which EYA3 may promote GC cell growth. In vivo experiments supported the in vitro findings, showing slower tumor growth with reduced EYA3 expression. Our study confirms the upregulation of EYA3 in GC and its association with poor prognosis. EYA3 promotes GC cell proliferation and tumor growth by activating the mTORC1 signaling pathway and inhibiting autophagy. These findings highlight the potential of EYA3 as a therapeutic target for GC, providing a foundation for future research and treatment strategies. Despite the promising data, the limitations of sample size and the need for further mechanistic studies are acknowledged.

MDM2 drives resistance to Osimertinib by contextually disrupting FBW7-mediated destruction of MCL-1 protein in EGFR mutant NSCLC.

Overcoming resistance to Osimertinib in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) is clinically challenging because the underlying mechanisms are not fully understood. The murine double minute 2 (MDM2) has been extensively described as a tumor promotor in various malignancies, mainly through a negative regulatory machinery on the p53 tumor suppressor. However, the significance of MDM2 on the sensitivity to Osimertinib has not been described. Osimertinib resistant cells were generated by standard dose escalation strategy and individual resistant clones were isolated for MDM2 testing. The MDM2 and its mutant constructs (ΔPBD, ΔRING, C464A) were introduced into PC-9, HCC827 and H1975 cells and evaluated for the sensitivity to Osimertinib by MTT assay, colony formation, EdU assay and TUNEL assay. MDM2 expression in resistant cells was manipulated by pharmacological and molecular approaches, respectively. Proteins that were implicated in PI3K/Akt, MAPK/Erk and apoptosis signaling were measured by Western blot analysis. Candidate proteins that interacted with MDM2 were captured by immunoprecipitation and probed with indicated antibodies. In comparison with parental PC-9 cells, the PC-9 OR resistant cells expressed high level of MDM2. Ectopic expression of MDM2 in PC-9, HCC827 and H1975 sensitive cells generated an Osimertinib resistant phenotype, regardless of p53 status. MDM2 promoted resistance to Osimertinib through a PI3K/Akt and MAPK/Erk-independent machinery, in contrast, MDM2 selectively stabilized MCL-1 protein to arrest Osimertinib-induced cancer cell apoptosis. Mechanistically, MDM2 acted as a E3 ligase to ubiquitinate FBW7, a well-established E3 ligase for MCL-1, at Lys412 residue, which resulted in FBW7 destruction and MCL-1 stabilization. Targeting MDM2 to augment MCL-1 protein breakdown overcame resistance to Osimertinib in vitro and in vivo. Finally, the clinical relevance of MDM2-FBW7-MCL-1 regulatory axis was validated in mouse xenograft tumor model and in NSCLC specimen. Overexpression of MDM2 is a novel resistant mechanism to Osimertinib in EGFR mutant NSCLC. MDM2 utilizes its E3 ligase activity to provoke FBW7 destruction and sequentially leads to MCL-1 stabilization. Cancer cells with aberrant MDM2 state are refractory to apoptosis induction and elicit a resistant phenotype to Osimertinib. Therefore, targeting MDM2 would be a feasible approach to overcome resistance to Osimertinib in EGFR mutant NSCLC.

Tumor heterogeneity and cooperating cancer hallmarks driven by divergent EMT programs.

Epithelial-to-mesenchymal transition (EMT) is known to play roles in orchestrating cellular plasticity across many physiological and pathological contexts. Partial EMT, wherein cells maintain both epithelial and mesenchymal features, is gaining recognition for its functional importance in cancer in recent years. There are many factors regulating both partial and full EMT, and the precise mechanisms underlying these processes vary depending on the biological context. Furthermore, how different EMT states cooperate to create a heterogeneous tumor population and promote different pro-malignant features remains largely undefined. In a recent study published in Nature Cancer, Youssef and colleagues described how two disparate EMT programs, active in either organ fibrosis or embryonic development, are utilized within different cells within the same murine mammary tumor model. This work provides mechanistic insight into the development of intratumoral heterogeneity, providing evidence for the cooperation between the two EMT trajectories.

Abstract I003: Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics

Abstract Over 100 modifications to RNA are known to occur in human cells, where they play critical roles in many aspects of normal cellular physiology, such as cell fate decisions and terminal differentiation, through effects on RNA biology such as protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes suggests many are cancer essential enzymes with striking synthetic lethal profiles, including the DHX9 helicase. DHX9 is a multifunctional DExH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops) and DNA/RNA G-quadruplexes. DHX9 interacts with and regulates many proteins, including key members of DNA damage repair pathways. We have found that DHX9 knockdown is synthetic lethal in microsatellite high (MSI-H) or defective mismatch repair (dMMR) tumor models. A suite of assays was developed to identify and optimize potent and selective inhibitors of the DHX9 helicase, which recapitulate our findings with genetic tools. Profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with mutations in the DNA damage repair genes BRCA1 and/or BRCA2 are also responsive to DHX9 inhibitor treatment in vitro and in vivo. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress, leading to cell cycle arrest and apoptosis. These results suggest that DHX9 inhibitors are a novel treatment modality for patients with defective DNA damage repair pathways such as dMMR and/or BRCA mutations. Citation Format: Serena J Silver. Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I003.

Abstract PR004: Second generation RNA Polymerase I inhibitor PMR-116 targets ribsomal RNA synthesis to potently treat a broad spectrum of malignancies

Abstract Purpose and rational for the study: The purpose of the study was to develop novel 2nd generation ribosomal RNA synthesis inhibitors for cancer treatment. Ribosome biogenesis (RiBi) is essential for cell growth and proliferation. Transcription of ribosomal RNA genes by Pol I is a critical step in RiBi and is tightly regulated by tumor suppressors and oncoproteins. Indeed, the ability of the potent oncoprotein, MYC, to drive RiBi is necessary for its oncogenic activity (Bywater et al., Cancer Cell, 2011). These data suggested that targeting RiBi, might be a promising strategy to treat malignancies, especially those driven by MYC. To test this, we developed the 1st small molecule inhibitor of Pol I transcription, CX-5461 (Drgyin et al., Cancer Research 2011). While CX-5461 has shown promising activity in pre-clinical studies (Bywater et al., Cancer Cell, 2011; Rebello et al., Clin Cancer Res., 2016; Hein et al., Blood, 2017) and in early Phase trials (Khot et al., Cancer Discovery, 2019; Hilton et al., Nat Commun. 2022) its mechanism of action remains controversial with evidence of off-target activity inducing DNA damage through inhibition of TOPO2a (Cameron et al., Biomedicines, 2024; Koh et al., Nature Genetics, 2024). Thus, to determine if selective inhibition of Pol I transcription in the absence of DNA damage can be used to therapeutical treat cancer in vivo, we developed a series of more selective 2nd generation Pol I inhibitors with our lead compound being PMR-116. Methods and summary of data: We demonstrate that PMR-116 is selective for inhibition of Pol I over Poll II transcription and functions by stalling the Pol I complex at the rDNA promoter. PMR-116, exhibits improved drug-like properties and pharmacokinetics compared to 1st generation CX-5461. As a single agent PMR-116 efficiently treats a range of pre-clinical models of hematologic and solid tumors including lymphoma, AML, hepatocellular carcinoma and prostate cancer. MYC expression positively correlates with sensitivity to PMR-116 in human cancer cell lines and high MYC tumors in vivo are exceptionally sensitive to PMR-116. In contrast to CX-5461, the therapeutic response to PMR-116 occurs in the absence of DNA damage suggesting that DNA damage is not an obligatory response to Pol I transcription inhibition but rather an off-target effect of the 1st generation drug. Small molecule drug combination screens in AML cells demonstrate that PMR-116 works synergistically to reduce cell viability with a range of drugs including carfilzomib and quisinostat used for treatment of hematologic malignancies. Conclusions: Our data establishes inhibition of RNA polymerase I as a bone fide therapeutic target for cancer therapy and demonstrated that PMR-116 is a promising new broad- spectrum anti-cancer drug particularly for those tumors driven by MYC, which are highly refractory to standard therapies. Based on these data we have launched a Phase I clinical trial of PMR-116 in patients with advanced solid tumors (CTRN12620001146987). Citation Format: Rita Ferreira, Katherine M. Hannan, Konstantin Panov, Thejanni Udumanne, Amee J George, Zaka Yuen, Perlita Poh, Eric Kusnadi, Alisee Huglo, Mitchell Lawrence, Mustapha Haddach, Denis Drygin, Luc Furic, Ross D Hannan, Nadine Hein. Second generation RNA Polymerase I inhibitor PMR-116 targets ribsomal RNA synthesis to potently treat a broad spectrum of malignancies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR004.

Stearoyl-CoA desaturase in CD4+ T cells suppresses tumor growth through activation of the CXCR3/CXCL11 axis in CD8+ T cells.

Within the tumor microenvironment, altered lipid metabolism promotes cancer cell malignancy by activating oncogenic cascades; however, impact of lipid metabolism in CD4+ tumor-infiltrating lymphocytes (TILs) remains poorly understood. Here, we elucidated that role of stearoyl-CoA desaturase (SCD) increased by treatment with cancer-associated fibroblast (CAF) supernatant in CD4+ T cells on their subset differentiation and activity of CD8+ T cells. In our study, we observed that CD4+ TILs had higher lipid droplet content than CD4+ splenic T cells. In tumor tissue, CAF-derived supernatant provided fatty acids to CD4+ TILs, which increased the expression of SCD and oleic acid (OA) content. Increased SCD expression by OA treatment enhanced the levels of Th1 cell markers TBX21, interleukin-2, and interferon-γ. However, SCD inhibition upregulated the expression of regulatory T (Treg) cell markers, FOXP3 and transforming growth factor-β. Comparative fatty acid analysis of genetically engineered Jurkat cells revealed that OA level was significantly higher in SCD-overexpressing cells. Overexpression of SCD increased expression of Th1 cell markers, while treatment with OA enhanced the transcriptional level of TBX21 in Jurkat cells. In contrast, palmitic acid which is higher in SCD-KO cells than other subclones enhanced the expression of Treg cell markers through upregulation of mitochondrial superoxide. Furthermore, SCD increased the secretion of the C-X-C motif chemokine ligand 11 (CXCL11) from CD4+ T cells. The binding of CXCL11 to CXCR3 on CD8+ T cells augmented their cytotoxic activity. In a mouse tumor model, the suppressive effect of CD8+ T cells on tumor growth was dependent on CXCR3 expression. These findings illustrate that SCD not only orchestrates the differentiation of T helper cells, but also promotes the antitumor activity of CD8+ T cells, suggesting its function in adverse tumor microenvironments.

Value of [68Ga]Ga-NYM046 PET/CT, in Comparison with 18F-FDG PET/CT, for Diagnosis of Clear Cell Renal Cell Carcinoma.

This study aimed to investigate the diagnostic efficacy of [68Ga]Ga-NYM046 PET/CT in animal models and patients with clear cell renal cell carcinoma (ccRCC) and to compare its performance with that of 18F-FDG PET/CT. Methods: The invivo biodistribution of [68Ga]Ga-NYM046 was evaluated in mice bearing OS-RC-2 xenografts. Twelve patients with ccRCC were included in the study; all completed paired [68Ga]Ga-NYM046 PET/CT and 18F-FDG PET/CT. The diagnostic efficacies of these 2 PET tracers were compared. Moreover, the positive rate of carbonic anhydrase IX in the pathologic tissue sections was compared with the SUVmax obtained by PET/CT. Results: The tumor accumulation of [68Ga]Ga-NYM046 at 1 h after injection in OS-RC-2 xenograft tumor models was 7.21 ± 2.39 injected dose per gram of tissue. Apart from tumors, the kidney and stomach showed high-uptake distributions. In total, 9 primary tumors, 96 involved lymph nodes, and 147 distant metastases in 12 patients were evaluated using [68Ga]Ga-NYM046 and 18F-FDG PET/CT. Compared with 18F-FDG PET/CT, [68Ga]Ga-NYM046 PET/CT detected more primary tumors (9 vs. 1), involved lymph nodes (95 vs. 92), and distant metastases (137 vs. 127). In quantitative analysis, the primary tumors' SUVmax (median, 13.5 vs. 2.4; z = -2.668, P = 0.008) was significantly higher in [68Ga]Ga-NYM046 PET/CT. Conversely, the involved lymph nodes' SUVmax (median, 5.9 vs. 7.6; z = -3.236, P = 0.001) was higher in 18F-FDG PET/CT. No significant differences were found for distant metastases (median SUVmax, 5.0 vs. 5.0; z = -0.381, P = 0.703). Higher [68Ga]Ga-NYM046 uptake in primary tumors corresponded to higher expression of carbonic anhydrase IX, with an R 2 value of 0.8274. Conclusion: [68Ga]Ga-NYM046 PET/CT offers a viable strategy for detecting primary tumors, involved lymph nodes, and distant metastases in patients with ccRCC.

Near-infrared fluorescent molecular probes with cetuximab in the in vivo fluorescence imaging for epithelial ovarian cancer.

Near-infrared fluorescence (NIRF) imaging is an excellent choice for image-guided surgery due to its simple operation and non-invasiveness. Developing tumor-specific fluorescent molecular probes is key to fluorescence imaging-guided surgery. EGFR (epidermal growth factor receptor) is closely related to the proliferation and growth of tumor cells and is highly expressed in epithelial ovarian cancer (EOC). The study aims to construct a NIR fluorescent molecular probe using cetuximab (an EGFR monoclonal antibody) and investigate its feasibility for targeting EOC in vivo through fluorescence imaging. We determined the expression of EGFR in EOC. NIR fluorescent molecular probe with cetuximab (cetuximab-Cy7) was chemically engineered and identified. The subcutaneous xenografted tumor model of EOC was induced using SKOV3-Luc cell line with positive expression of EGFR. Cetuximab-Cy7 was used for in vivo fluorescence imaging, and phosphate-buffered saline, free Cy7 dye and mouse isotype immunoglobulin G-Cy7 were used as controls. NIRF imaging system was performed to study the distribution and targeting of the probes. Tumors were imaged in situ and ex vivo, and fluorescent intensity was quantified. Resected specimens were analyzed to confirm diagnosis, and immunohistochemical (IHC) staining was used to identify EGFR expression. EGFR expression was increased in EOC tissues than fallopian tube tissues. The high expression of EGFR was significantly correlated with well-differentiation, residual lesions ≤ 1cm, no recurrence and increased survival. NIRF imaging showed that the cetuximab-Cy7 enabled detection of tumor lesions in EOC-bearing mice with the optimal dose of 30µg. The suitable imaging time window may be 24-96h post-injection. Ex vivo fluorescence imaging indicated that fluorescent signal was mainly detected in the tumor and the lung. IHC results confirmed that xenografts were EGFR positive. Cetuximab-Cy7 can specifically target the tumors of EOC xenografted nude mice. This research lays the foundation for future studies on EOC surgery navigation.