Expression In Tumor Cells Research Articles

Impact of HIF-1α, LOX and ITGA5 Synergistic Interaction in the Tumor Microenvironment on Colorectal Cancer Prognosis

Background: As colorectal cancers are histopathologically and molecularly highly heterogeneous tumors, it is necessary to consider the tumor’s microenvironment as well as its cellular characteristics in order to determine the biological behavior of the tumor. This study included 100 patients who underwent resection for colorectal cancer. We aimed to investigate the relationships between the expression status of the HIF-1α, LOX and ITGA5 proteins and clinicopathologic parameters. Methods: HIF-1α, LOX and ITGA5 antibodies were applied immunohistochemically to tissue microarrays prepared from tumor samples. Expression status in the tumor microenvironment were evaluated using a combined scoring system based on staining intensity and the percentage of positively stained cells. Nuclear HIF-1α expression in tumor cells was quantified, with >1% considered positive. The staining of HIF-1α, ITGA5 and LOX was analyzed in relation to prognostic and molecular features. Results: The staining of HIF-1α, ITGA5 and LOX in the tumor microenvironment demonstrated a positive correlation with one another and with HIF-1α and LOX expression in tumor cells. In patients with KRAS, NRAS or BRAF mutation and the moderate to strong expression of all three of these proteins in the tumor microenvironment, the number of metastatic lymph nodes was higher than in other patients. Stage IV patients with the moderate to strong expression of HIF-1α, ITGA5 or LOX in the microenvironment had lower progression-free survival than those with weak expression (p < 0.05). In addition, female gender; moderate to strong HIF-1α, LOX and ITGA5 stromal expression; and metastatic first line chemotherapy only were found to be independently associated with an increased risk of progression. Conclusion: These markers may be useful in predicting treatment responses and may also guide the development of alternative or combined treatments that specifically target molecules such as HIF and LOX. Our study should be supported by more comprehensive studies addressing the tumor stroma and its prognostic importance.

Clonorchis sinensis Promotes Intrahepatic Cholangiocarcinoma Progression by Activating FASN-Mediated Fatty Acid Metabolism.

Clonorchis sinensis infection is an important risk factor for intrahepatic cholangiocarcinoma (ICC). C. sinensis positive (C.s+) ICC patients had much shorter overall survival (OS) compared with C. sinensis negative (C.s-) group. This study aims to explore the impact and underlying mechanism of C. sinensis infection on ICC progression. In this study, ICC patients underwent surgery from two medical centers enrolled. RNA sequencing was used to determine the downstream activated pathways and genes. Furthermore, we demonstrated the potential mechanism of C. sinensis infection in promoting ICC progression through invitro co culture systems and two animal models. Through RNA sequencing, we found fatty acid metabolism and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly elevated in C.s+ ICCs. Then, we found excretory/secretory products (ESPs) secreted by C. sinensis could significantly upregulate the expression of transcription factor E2F1, thereby promoting FASN expression and fatty acid synthesis in tumor cells, which ultimately accelerating tumor progression. However, the promotive effect disappeared when FASN was knocked down. Meanwhile, ESPs could promote tumor growth, increasing FASN expression and free fatty acid level in both subcutaneous and orthotopic mouse models. This study indicates that C. sinensis infection could upregulate the level of FASN and activate fatty acid synthesis pathway, thereby accelerating ICC progression. This provides a new insight for the clinical treatment of ICC with C. sinensis infection.

Comparative evaluation of PD-L1 expression and tumor immune microenvironment in molecular subtypes of muscle-invasive bladder cancer and its correlation with survival outcomes.

Immune checkpoint inhibitors have revolutionized treatment of platinum-refractory advanced bladder cancer, offering hope where options are limited. Response varies, however, influenced by factors such as the tumor's immune microenvironment and prior therapy. Muscle-invasive bladder cancer (MIBC) is stratified into molecular subtypes, with distinct clinicopathologic features affecting prognosis and treatment. This study assessed the expression of programmed cell death 1 ligand 1 (PD-L1) and other immune markers in MIBC, categorized by molecular phenotype. Using GATA3 and CK5/6 immunohistochemistry, 90 neoadjuvant chemotherapy-naive MIBC cases were classified into luminal andnon-luminalsubtypes. The immune microenvironment was characterized through immunostaining for PD-L1, CD4, and CD8. We applied PD-L1 positivity thresholds of 1% or greater for tumor cells and 5% or greater for immune cells. Tumors were examined for PD-L1 expression, histologic subtypes, and immune cell infiltration. Varied expression of PD-L1 and T-cell subtype densities were observed among MIBC subtypes. The double-negative subtype displayed the highest PD-L1 immune cell expression and stromal CD4 and CD8 T-cell densities, indicating an active immune profile. The basal subtype exhibited the highest PD-L1 positivity in tumor cells. In contrast, the luminal type showed the lowest PD-L1 tumor and immune cell expression, with high intratumoral CD4 T-cell density. Although PD-L1 expression in tumor or immune cells did not independently affect survival, patients with basal and double-negative tumors had poorer overall survival. This study highlighted the immune diversity of MIBC in the context of molecular subtypes. Distinct molecular and immune profiles could guide the development of predictive signatures for enhanced immunotherapy response in advanced bladder cancer.

Ginsenoside RK3 inhibits glioblastoma by modulating macrophage M2 polarization via the PPARG/CCL2 axis

BackgroundGlioblastoma is recognized as the most aggressive form of intracranial tumor, presenting significant challenges in treatment. Recent emphasis has been placed on the potential of traditional Chinese medicine (TCM) as an adjuvant treatment for cancer. Methods: We employed a series of assays—including CCK8, EdU, Transwell, and neurosphere formation—to evaluate the impact of ginsenoside RK3 on the phenotype of GBM. The modulation of macrophage M2 polarization by ginsenoside RK3 was assessed through flow cytometry, immunohistochemistry, and Western blot analysis. Furthermore, we utilized sequencing analysis and network pharmacology to identify potential therapeutic targets. ResultsOur findings reveal that ginsenoside RK3 not only inhibits the phenotype of glioblastoma cells but also suppresses tumor progression in vivo while attenuating macrophage M2 polarization within the tumor immune microenvironment. Notably, ginsenoside RK3 down-regulates PPARG expression in tumor cells, leading to decreased secretion of CCL2, which subsequently diminishes macrophage M2 polarization. Additionally, we demonstrated that combining ginsenoside RK3 with temozolomide significantly enhances the inhibition of glioblastoma's malignant characteristics and progression. ConclusionsThis study innovatively highlights the dual mechanism of ginsenoside RK3 in glioblastoma treatment: it impedes tumor progression by modulating the PPARG/CCL2 pathway and enhances the efficacy of temozolomide. Our research underscores the promising role of herbal medicine in the management of glioblastoma, paving the way for novel therapeutic strategies that integrate traditional approaches with conventional treatments.

Distribution and clinicopathological characteristics of G-CSF expression in tumor cells and stromal cells in upper tract urothelial carcinoma

Abstract Background Urothelial carcinoma (UC) is a common type of malignant disease; however, the diagnostic and prognostic markers of upper urinary tract urothelial cancer (UTUC) remain poorly understood because of its rarity. Methods To clarify the clinicopathological significance of granulocyte-colony stimulating factor (G-CSF) in UTUC, we analyzed the expression and distribution of G-CSF in 112 upper tract urothelial carcinoma (UTUC) samples with immunohistochemistry. Results In normal urothelium, G-CSF expression was weak or absent, whereas high expression of G-CSF was observed in UTUC tissues, both in tumor cells (TCs) and stromal cells (SCs). G-CSF expression in the TCs and SCs was associated with nodular/flat morphology, high grade, advanced T stage, and lymphovascular invasion in UTUC. G-CSF expression in SCs was associated with poor prognosis and was an independent prognostic factor. Public data showed that G-CSF expression was also associated with decreased progression-free survival and disease-specific survival. A prognostic model was constructed by incorporating the presence or absence of G-CSF expression along with clinicopathologic factors, which allowed for a more accurate prediction of poor prognosis. We further showed that G-CSF expression was associated with a high Ki-67 labeling index and with PD-L1, HER2, and p53 expression in UTUC. Conclusion G-CSF expression in TCs and SCs may play a crucial role in UTUC tumor progression. Notably, stromal G-CSF expression showed significant prognostic value, even when compared to major clinicopathological factors, suggesting that the evaluation of G-CSF expression may contribute to clinical decision-making in patients with UTUC.

Chemokine CXCL12 Activates CXC Receptor 4 Metastasis Signaling Through the Upregulation of a CXCL12/CXCR4/MDMX (MDM4) Axis.

The metastasis-promoting G-protein-coupled receptor CXC Receptor 4 (CXCR4) is activated by the chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1). The CXCL12/CXCR4 pathway in cancer promotes metastasis but the molecular details of how this pathway cross-talks with oncogenes are understudied. An oncogene pathway known to promote breast cancer metastasis in MDA-MB-231 xenografts is that of Mouse Double Minute 2 and 4 (MDM2 and MDM4, also known as MDMX). MDM2 and MDMX promote circulating tumor cell (CTC) formation and metastasis, and positively correlate with a high expression of CXCR4. Interestingly, this MDMX-associated upregulation of CXCR4 is only observed in cells grown in the tumor microenvironment (TME), but not in MDA-MB-231 cells grown in a tissue culture dish. This suggested a cross-talk signaling factor from the TME which was predicted to be CXCL12 and, as such, we asked if the exogenous addition of the cell non-autonomous CXCL12 ligand would recapitulate the MDMX-dependent upregulation of CXCR4. We used MDA-MB-231 cells and isolated CTCs, with and without MDMX knockdown, plus the exogenous addition of CXCL12 to determine if MDMX-dependent upregulation of CXCR4 could be recapitulated outside of the TME context. We added exogenous CXCL12 to the culture medium used for growth of MDA-MB-231 cells and isogenic cell lines engineered for MDM2 or MDMX depletion. We carried out immunoblotting, and quantitative RT-PCR to compare the expression of CXCR4, MDM2, MDMX, and AKT activation. We carried out Boyden chamber and wound healing assays to assess the influence of MDMX and CXCL12 on the cells' migration capacity. The addition of the CXCL12 chemokine to the medium increased the CXCR4 cellular protein level and activated the PI3K/AKT signaling pathway. Surprisingly, we observed that the addition of CXCL12 mediated the upregulation of MDM2 and MDMX at the protein, but not at the mRNA, level. A reduction in MDMX, but not MDM2, diminished both the CXCL12-mediated CXCR4 and MDM2 upregulation. Moreover, a reduction in both MDM2 and MDMX hindered the ability of the added CXCL12 to promote Boyden chamber-assessed cell migration. The upregulation of MDMX by CXCL12 was mediated, at least in part, by a step upstream of the proteasome pathway because CXCL12 did not increase protein stability after cycloheximide treatment, or when the proteasome pathway was blocked. These data demonstrate a positive feed-forward activation loop between the CXCL12/CXCR4 pathway and the MDM2/MDMX pathway. As such, MDMX expression in tumor cells may be upregulated in the primary tumor microenvironment by CXCL12 expression. Furthermore, CXCL12/CXCR4 metastatic signaling may be upregulated by the MDM2/MDMX axis. Our findings highlight a novel positive regulatory loop between CXCL12/CXCR4 signaling and MDMX to promote metastasis.

HPV frequency in oral cavity and oropharynx cancers and its association with immune checkpoint inhibitors

Objectives: The prevalence of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinomas (SqCCs) varies ethnically and geographically and has better prognostic features compared to HPV-negative cases. Escape from the immune system is one of the important mechanisms in the development of oropharyngeal SCC. The interaction of programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) causes T cell suppression. In our study, we aimed to evaluate the frequency of HPV and its relation with PD-:PD-L1, which is closely related to lymphoid tissue and important in prognosis and treatment in oral cavity and oropharynx SCCs. Method: We retrospectively investigated the presence of HPV in sections prepared from paraffin blocks of biopsy and excision materials by immunohistochemical staining method with p16 antibody and HPV in-situ hybridization (ISH) method in our series of 70 patients diagnosed with oral (SqHC). We also evaluated PD-L1 expression in tumor cells and PD-1 expression in lymphoid cells in the tumor microenvironment by immunohistochemical staining method. Results: HPV positivity was detected in three tumors, two in the oropharynx and one lateral to the tongue. PD-L1 positivity was observed in 45 (64.3%) cases. PD-1 expression was moderate or severe in 75.6% of PD-L1 positive cases and 24% of PD-L1 negative cases and this ratio was statistically significant. PD-L1 positivity was detected in only one of the 3 HPV positive cases. There was no significant difference in overall survival in PD-L1 positive patients compared to PD-L1 negative ones. Discussion: PD-1 or PD-L1 expression can be observed in oral cavity and oropharynx SCC regardless of HPV status. Although HPV positivity is frequently reported to be associated with good prognosis, studies are limited especially in our country. The prognostic and predictive importance of these biomarkers should be supported by more comprehensive studies.

Therapeutic Potential of IL‐37 in Cervical Cancer: Suppression of Tumour Progression and Enhancement of CD47‐Mediated Macrophage Phagocytosis

ABSTRACTAs a promising therapeutic approach, immunotherapy is being extensively investigated in cervical cancer. Although immunotherapy has been validated to improve progression‐free survival and overall survival in clinical trials, the overall response rate for cervical cancer remains inadequate, necessitating further improvement. Interleukin (IL)‐37, an emerging immunomodulator, exhibits antitumour potentials by inhibiting tumour progression and regulating tumour‐associated macrophage recognition. We found a significant downregulation of IL‐37 expression in cervical cancer, correlated with a poor prognosis. Moreover, the upregulation of IL‐37 expression exhibited a suppressive effect on various tumorigenic processes, suppressing the proliferation, invasion, migration, apoptosis and angiogenesis of tumour cells. We also found that the upregulation of IL‐37 suppressed cluster of differentiation 47 (CD47) expression in tumour cells via suppression of the signal transducer and activator of transcription 3 (STAT3) expression and phosphorylation, thereby enhancing macrophage recognition and phagocytosis to tumour cells, ultimately reducing immune evasion. Overall, our study highlighted the crucial role of IL‐37 in antitumour efficacy and downregulating the expression of CD47 in tumour cells to reduce immune evasion, suggesting the potential of IL‐37 as a prognostic biomarker in cervical cancer and offering innovative therapeutic strategies to improve cancer treatment outcomes.

Elevated expression of REV7 correlates with poor prognosis in lung adenocarcinoma and its inactivation in carcinoma cells enhances chemosensitivity.

REV7 is a multifunctional protein involved in the DNA damage response, cell cycle regulation, gene expression, or primordial germ cell maintenance. REV7 expression in tumor cells is associated with clinical aggressive features and chemoresistance in several human malignancies, however, the clinicopathological significance of REV7 in lung adenocarcinoma (LUAD) has not been studied yet. In this study, we investigated the significance of REV7 expression in LUAD using clinical materials and cell lines. REV7 expression in 142 invasive LUADs were determined using immunohistochemistry, and the relationship between REV7 expression and clinicopathological features was analyzed. High levels of REV7 expression in tumor tissues were positively associated with progressive tumor behavior as assessed by Ki-67 labeling indexes (p < 0.001), maximum standardized uptake values on positron emission tomography (p = 0.005), pathological stage (p = 0.031), N factor (p = 0.048), recurrence (p = 0.038), and disease-specific death (p = 0.020). The REV7-high-expression group showed poorer relapse-free survival (RFS) (p = 0.025) and overall survival (OS) (p = 0.019) compared to the REV7-low-expression group, and REV7 was a significant prognostic factor for RFS and OS. CRISPR/Cas9-mediated REV7-knockout and siRNA-mediated REV7 knockdown were carried out using the LUAD cell lines A549 and H1975, respectively, and it was demonstrated that REV7 inactivation led to slower cell growth, attenuated activation of AKT signaling, and enhanced chemosensitivity compared with control cells. These results suggest that REV7 is a potential predictive biomarker for poor prognosis in invasive LUAD and a possible molecular target for LUAD management.

A multifunctional nanoplatform capable of dual action on tumor PD-L1 for enhanced cancer theranostics

Due to the large amount of PD-L1 on the surface of tumor cells, antibody drugs can only block a small part of PD-L1, limiting the effectiveness of immunotherapy. Here, a MnS nanocluster grafted with dimeric PD-L1 affibody (ZPD-L1) was developed to double act on PD-L1 of tumor cells. ZPD-L1 can specifically target PD-L1 on the surface of tumor cells, and block the interaction between PD-1 and PD-L1 to a certain extent. In acidic tumor microenvironment, MnS nanoclusters can release Mn2+ and H2S. The released Mn2+ can induce reactive oxygen species (ROS) generation through Fenton-like reaction, and serve as magnetic resonance imaging (MRI) contrast agent for effective therapeutic monitoring. H2S can down-regulate PD-L1 expression of tumor cells by amplifying ROS production to inhibit ATP level. Based on ZPD-L1-mediated PD-1/PD-L1 blocking and ROS-induced downregulation of PD-L1 expression, MnS nanoclusters can achieve dual action on tumor PD-L1, amplify immunogenic cell death, maximize the activation of anti-tumor immune response and inhibit tumor growth.

Immuno-oncologic profiling by stage-dependent transcriptome and proteome analyses of spontaneously regressing canine cutaneous histiocytoma.

Canine cutaneous histiocytoma (CCH) is a tumor that originates from dermal Langerhans cells and affects particularly young dogs. The common spontaneous regression of CCH makes it an interesting model in comparative oncology research. Previous studies have indicated that anti-tumor immune responses may be involved, but details remain speculative to date. Here, we asked which specific immuno-oncological dynamics underlie spontaneous regression of CCH on mRNA and protein levels. QuantSeq 3' mRNA sequencing with functional over-representation analysis and an nCounter RNA hybridization assay were employed on 21 formalin-fixed, paraffin-embedded CCH samples representing three different tumor stages (dataset information: GSE261387-Immuno-Oncologic Profiling by Stage-Dependent Transcriptome and Proteome Analyses of Spontaneously Regressing Canine Cutaneous Histiocytoma-OmicsDI). Nine additional samples were subjected to matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Surprisingly, only minor stage-specific differences were found. When we investigated expression of B7 family ligands and CD28 family receptors holding co-stimulatory and -inhibitory functions, respectively, we found a higher abundance of CD80, CD86, CTLA4 and CD28, which may trigger a balanced activation of lymphocyte-mediated immune responses. CD80 and CD86 expressing cells were further quantified by in situ hybridization and compared with data from three cases of canine histiocytic sarcoma (HS), a malignant tumor variant originating from antigen-presenting interstitial dendritic cells. A stage-specific increase of CD80 expressing cells was recorded in CCH from the tumor bottom to the top, while CD86 was continuously and homogenously expressed at high levels. Overall expression of CD80 in CCH was similar to that in HS (73.3 ± 37.4% vs 62.1 ± 46.4%), while significantly more CD86 expressing tumor cells were found in CCH (94.7 ± 10.3%) when compared to HS (57.6 ± 11.0%). Our data suggest that major immuno-oncological pathways are not regulated during regression of CCH on the mRNA or protein levels as detectable by the methods used. Instead, our data provide further evidence supporting previous hypotheses towards a role of immune stimulatory B7 family ligands and CD28 family receptors in the regression of CCH.

Enhancing antitumor immunity in Lewis lung cancer through plasma-treated medium-induced activation of dendritic cells

BackgroundRecently, atmospheric non-thermal plasma jet-treated medium (PTM) has been recognized as a novel strategy in cancer therapy and lymphocyte activation. However, PTM has limitations in inducing a robust antitumor-immune response. This study demonstrated that PTM treatment inhibited tumor progression by activating dendritic cells (DCs).MethodIn this study, we investigated the effects of PTM on selective cytotoxicity and intracellular reactive oxygen species (ROS) generation and oxidative stress-mediated signaling (e.g., glutathione peroxidase, catalase) using respective fluorescence probes in Lewis lung cancer (LLC) cells. Then, the PTM affects the expression of interferon-gamma (IFN)-γ-induced programmed death-ligand 1 (PD-L1) and inhibition of signal transducer and activator of transcription 1 (STAT1) in LLC cells using immunoblotting. Additionally, PTM effects on the tumor cell’s death and activation of DCs were done by co-culturing DCs with or without tumor cells. Further, a mouse model was used to evaluate the synergistic antitumor effects of PTM and DCs where tumors are grown under the skin.ResultsPTM-exposed tumor cells increase intracellular superoxide production, enhancing ROS generation and leading to cancer immunogenic cell death. In addition, PTM suppresses IFN-γ-induced PD-L1 expression and STAT1 activation in tumor cells. The activation of DCs induced by PTM is downregulated when these cells are co-cultured with tumor cells. In vivo, intraperitoneal injection of PTM-activated DCs, as a synergistic agent to intertumoral PTM treatment, led to increased CD4+ and CD8+ T cell infiltration into the tumor and spleen and eventually decreased tumor growth.ConclusionOverall, this research introduces a promising avenue for improving lung cancer treatment using PTM to stimulate an immune response and induce cell death in tumor cells. Further studies will be essential to validate these findings and explore clinical applications.Graphical

Progress in PGK1 in Prostate Cancer

Phosphoglycerol kinase 1 (PGK1) is the first key metabolic enzyme that produces ATP during glycolysis and is involved in the glycolytic pathway of prostate cancer. PGK 1 can not only act as a metabolic enzyme to affect tumor growth, but also affect the gene expression, energy metabolism, molecular regulation and other processes of tumor cells through its non-metabolic enzyme function, and then mediate the growth, migration and invasion of tumors, and aggravate the biological characteristics of malignant cancer cells. In this review, we reviewed the structure and function of PGK1 and its relationship with prostate cancer to clarify the important role of PGK1 in the progression of advanced adenoma and provide a theoretical basis for targeting PGK1 for drug development.

Multi-omics analysis reveals the role of the autophagy-related gene AGT in chemotherapy resistance in colorectal cancer and the therapeutic potential of its inhibitors.

Autophagy is a crucial mechanism for maintaining cellular homeostasis and responding to environmental stress, and it is closely linked to tumor drug resistance. Through multi-omics analysis, this study explores the expression patterns, functions, and potential role of the autophagy-related gene Angiotensinogen (AGT) in colorectal cancer (CRC), particularly in relation to chemotherapy resistance. This study first compared AGT expression between CRC and normal tissues using the GTEx and TCGA databases. Differences in expression were assessed using Wilcoxon Rank Sum Tests, and the prognostic impact of AGT was evaluated through univariate Cox survival analysis and meta-analysis. Functional enrichment was performed using the limma and fgsea packages. Drug sensitivity analysis was conducted based on the CTRP database, while immune infiltration was assessed using the CIBERSORT and ESTIMATE methods. Spatial transcriptomic characteristics were explored through 10x Visium technology and deconvolution analysis to investigate the correlation between AGT expression levels and tumor cell content.scRNA-seq data from CRC tissues were sourced from Tumor Immune Single Cell Hub (TISCH).Functional annotation was performed with Single-sample gene set enrichment analysis (SSGSEA), and pseudotime analysis using Monocle 2 mapped their developmental trajectories. The potential of AGT inhibitors in the treatment of CRC was analyzed using drug-target Mendelian randomization.Finally, Phenome-Wide Association Study (PheWAS) was conducted to evaluate genetic associations and potential side effects of AGT inhibitors. AGT expression was significantly higher in CRC tissues compared to normal tissues and was associated with shorter recurrence-free survival (RFS). Autophagy signaling pathways were markedly enriched in the high AGT expression group. AGT expression was positively correlated with resistance to chemotherapeutic agents such as gemcitabine, cisplatin, paclitaxel, and 5-fluorouracil. Spatial transcriptomic analysis revealed that AGT was predominantly expressed in malignant tumor regions. Single-cell analysis identified 21 distinct cell subpopulations across 13 major types. AGT expression was significantly higher in tumor samples, especially in the fibroblast C6 subpopulation. Tumor-related pathways were enriched in C1, C5, C6, and C8 subpopulations. Pseudotime analysis revealed that these subpopulations, particularly C6, were in terminal developmental stages.Drug-target Mendelian randomization analysis indicated a negative causal relationship between AGT inhibitors and the risk of both heart failure(ORdrug = 0.950, 95% CI, 0.912-0.990; P = 0.014) and CRC(ORdrug = 0.874, 95% CI: 0.792-0.964; P = 0.007).PheWAS analysis showed no genetic associations between AGT inhibitors and other traits, indicating its specificity and low risk of side effects. Elevated AGT expression in CRC is associated with resistance to chemotherapy, and its inhibition may offer a therapeutic avenue for the treatment of colorectal cancer.

Abstract C034: Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors

Abstract Although various effective anti-cancer treatments have become available over the last decades, resistance to all available therapies remains the major cause of death of cancer patients with disseminated tumors. Striking examples are patients with triple-negative breast cancer (TNBC), which are frequently defective in the repair of DNA double strand breaks, e.g. due to loss of BRCA1 function. Because of this defect, the patients initially respond very well to DNA damage-inducing chemotherapy. Unfortunately, disseminated tumors are usually not eradicated and resistant tumor cells are eventually selected from residual primary or metastatic tumor sites. It is therefore crucial to understand the molecular mechanisms underlying the drug tolerance of the residual tumor cells. To study residual disease, we used the K14cre;Brca1 F/F ;p53 F/F (KB1P) mouse model for hereditary breast cancer, which provides the unique opportunity to explore and target those mechanisms in a fully immunocompetent model. The mammary tumors that spontaneously develop highly resemble their human counterparts, both morphologically and in their therapy response. For example, tumors shrink in response to poly(ADP-ribose) polymerase inhibition, platinum-based treatment or the commonly used doxorubicin, docetaxel and cyclophosphamide (TAC) combination therapy. But despite repeated drug sensitivity, the KB1P mammary tumors are not eradicated, not even by a frequent dosing schedule. By combining single-cell RNA sequencing, spatial transcriptomics and imaging mass cytometry, we dissected the intratumoral heterogeneity and alterations in the tumor microenvironment of residual disease. We identified specific subpopulations of tumor cells that have a survival benefit after treatment, and these occurred together with an altered microenvironment characterized by a highly reactive stroma infiltrated with both anti-inflammatory and pro-tumoral immune cells. Interestingly, those structural and transcriptional changes are reversed in the relapsed tumors, highlighting the plasticity of drug tolerance. To investigate the mechanisms of the altered tumor-stroma interactions that are relevant for drug tolerance, we have designed a custom-made CRISPR/Cas9 library based on differential gene expression of the residual tumor cells. This library enables us to functionally test relevant mechanisms of drug tolerance in vivo and we expect that these analyses will provide useful insights into the tumor-stroma interactions that contribute to residual disease. Taken together, our detailed analyses demonstrate the substantial remodeling of tumor cells in their microenvironment upon treatment. To develop new therapeutic approaches to eradicate drug-tolerant tumor cells and thereby circumvent tumor relapse, it is essential to better understand the heterogeneous cellular composition of residual tumors in its spatial context. With this project, we hope to provide comprehensive data to develop better therapeutic strategies that target the tumor-stroma interaction and eradicate residual tumors. Citation Format: Morgane Decollogny, Demeter Túrós, Astrid Chanfon, Myriam Siffert, Ismar Klebic, Sven Rottenberg. Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary 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 C034.

Prevention of liver metastasis via the pharmacological suppression of AMIGO2 expression in tumor cells

AMIGO2 adheres to liver endothelium and induces liver metastasis. We revealed that genetically altering AMIGO2 expression in tumor cells affects their liver metastatic potential, depending on the AMIGO2 expression level. The aim of this study was to prevent liver metastasis by pharmacologically suppressing AMIGO2 expression. For screening, we used the mouse LV12 cells because of their affinity to adhere to liver endothelium and metastasize the liver owing to elevated AMIGO2 expression. Of the 285 compounds tested, 17 reduced AMIGO2 mRNA expression. We subsequently screened for compounds that inhibited tumor cell adhesion to liver endothelium and identified five compounds that inhibit three signaling pathways (MEK, JAK, and JNK). Treatment with these compounds inhibited liver metastasis of LV12 cells. Next, we used clinically available signal inhibitors (MEK inhibitor trametinib, JAK inhibitor ruxolitinib, and JNK inhibitor SP600125), and found that ruxolitinib inhibits AMIGO2 expression more stably. Furthermore, ruxolitinib inhibited the adhesion of LV12 cells to liver endothelium and suppressed liver metastasis. Using the MKN45 gastric cancer cells, we confirmed that ruxolitinib could prevent liver metastasis of human cancer cells. These results demonstrate that pharmacological inhibition of AMIGO2 expression in tumor cells is a promising novel strategy to prevent and control liver metastasis.

Cancer Immunotherapy Using AIRE Conditioning of the Tumor Epitopeome.

T cell immune tolerance is established in part through the activity of the Auto-immune Regulator (AIRE) transcription factor in the medullary Thymic Epithelial Cells (mTEC) of the thymus. AIRE induces expression of SELF peripheral tissue-specific antigens for presentation to naïve T cells to promote activation/deletion of potentially autoreactive T cells. We show, for the first time to our knowledge, that tumors mimic the role of AIRE in mTEC to evade immune rejection. Thus, by expressing a broad range of SELF epitopes against which minimal functional T cell reactivities exist because of thymic deletion, AIRE acts as a master controller of SELFNESS, effectively cloaking the tumor from T cell attack. Moreover, we describe a completely novel immunotherapy in which engineered changes in AIRE expression in tumor cells alters their profile of SELFNESS, exposing both AIRE-modified, and parental unmodified, tumor cells to T cell attack. Consistent with our studies, patient RNAseq shows expression of AIRE predicts response to immune therapies with a strong correlation between AIRE expression and markers of TCR signaling. Therefore, by re-setting the immunological SELFNESS of cancer cells, this novel AIRE-mediated immunotherapy 1). converts a highly tolerized T cell compartment into a heteroclitic tumor-reactive T cell population; 2) confers de novo sensitivity to immune checkpoint blockade upon non-immunogenic tumors; 3). completely removes the need to identify potentially immunogenic tumor-associated antigens as targets for generation of de novo CD8+ and helper CD4+ T cell responses; and 4) leads to potent T cell-mediated rejection of aggressive, immunologically cold, non-immunogenic tumors.

VisRNA: Interactive web server for scRNA-seq data analysis to discover therapeutic targets for non-small cell lung cancer

Abstract. Motivation: Non-small cell lung cancer (NSCLC) is a leading cause of lung cancer diagnoses and mortality worldwide. Single-cell RNA sequencing (scRNA-seq) has transformed our molecular understanding of cancer by revealing gene dysregulation in individual cells. This granularity allows the discovery of new therapeutic targets, promising NSCLC treatment strategies. Method: The study introduces VisRNA, a Python-based web application for analyzing scRNA-seq data statistically and functionally. VisRNA uses advanced machine learning algorithms to reduce multiple dimensionalities in scRNA-seq data and automatically annotate cell types. This method allows gene expression analysis across NSCLC tumor cell populations. Results: VisRNA analysis of NSCLC scRNA-seq datasets identified 14 distinct cell types with distinct gene expression patterns. VisRNA found several significantly differentially expressed genes in these cell populations that could be therapeutic targets. The application ranked drug candidates by molecular docking simulation performance, indicating potential efficacy against targets. Conclusion: VisRNA is a powerful tool for identifying new therapeutic targets and drug candidates for NSCLC, highlighting a significant advancement in scRNA-seq data analysis. VisRNA helps understand the diseases molecular basis by examining gene expression in individual NSCLC tumor cells, which may lead to better treatments.

Nuclear PD-L1 compartmentalization suppresses tumorigenesis and overcomes immunocheckpoint therapy resistance in mice via histone macroH2A1.

Canonically PD-L1 functions as the inhibitory immune checkpoint on cell surface. Recent studies have observed PD-L1 expression in the nucleus of cancer cells. But the biological function of nuclear PD-L1 (nPD-L1) in tumor growth and antitumor immunity is unclear. Here we enforced nPD-L1 expression and established stable cells. nPD-L1 suppressed tumorigenesis and aggressiveness in vitro and in vivo. Compared with PD-L1 deletion, nPD-L1 expression repressed tumor growth and improved survival more markedly in immunocompetent mice. Phosphorylated AMPKα (p-AMPKα) facilitated nuclear PD-L1 compartmentalization and then cooperated with it to directly phosphorylate S146 of histone variant macroH2A1 (mH2A1) to epigenetically activate expression of genes of cellular senescence, JAK/STAT, and Hippo signaling pathways. Lipoic acid (LA) that induced nuclear PD-L1 translocation suppressed tumorigenesis and boosted antitumor immunity. Importantly, LA treatment synergized with PD-1 antibody and overcame immune checkpoint blockade (ICB) resistance, which likely resulted from nPD-L1-increased MHC-I expression and sensitivity of tumor cells to interferon-γ. These findings offer a conceptual advance for PD-L1 function and suggest LA as a promising therapeutic option for overcoming ICB resistance.

Clinicopathological Characteristics of Neutrophil-Rich Hepatocellular Carcinoma: An Uncommon Subtype of Primary Liver Cancer.

Introduction. Neutrophil-rich hepatocellular carcinoma (HCC) is an extremely uncommon subtype of HCC with an overall incidence of <1%. Neutrophil-rich HCC shows poor cellular differentiation and sarcomatoid transformation in most patients. There is prominent neutrophilic inflammatory cell infiltration in the tumor. These tumors are associated with poor prognosis, high rate of recurrence, and metastasis. Methods. Herein, we investigated 4 patients with neutrophil-rich HCC reported at our center. Clinical, radiological, and pathological findings were reviewed. Immunophenotypic characterization of the tumors were done. Granulocyte colony-stimulating factor (G-CSF), programmed cell death ligand 1 (PD-L1), and mismatch repair immunostains were performed in all 4 tumors. Results. We report 4 neutrophil-rich HCCs in 3 male patients and one female patient with an age range of 43 to 64 years. Three underwent living donor liver transplantation and one underwent right hepatectomy. Tumor measured 0.5 cm to 12 cm in maximum dimension. Histologically, tumors demonstrated moderate to marked cellular pleomorphism. Spindle cell transformation was noted in 3 tumors. Three tumors showed vascular invasion, and one tumor showed bile duct invasion. Immunopositivity for Hep Par-1, arginase-1, and glypican-3 was present in all tumors. Tumors also expressed stemness markers including KRT19 and EpCAM. Cytoplasmic positivity for G-CSF and immunoexpression of PD-L1 was demonstrated. We also report proficient mismatch repair by immunohistochemistry in all tumors. Conclusion. Neutrophil-rich HCC is an aggressive primary liver cancer which demonstrates stemness-related features. Programmed cell death ligand 1 expression in tumor cells suggests distinct immunogenic features and potential role of anti-PD-L1 therapies in inoperable disease.