Human Tumor Research Articles

M6A-modified exosome-derived circHIF1α binding to KH domain of IGF2BP3 mediates DNA damage and arrests G1/S transition phase to resists bacterial infection in bacteremia

BackgroundAnimal and human health are seriously threatened by bacterial infections, which can lead to bacteremia and extremely high rates of morbidity and mortality. Recently, there have been reports indicating the involvement of exosomal circular RNAs (circRNAs) in a range of human disorders and tumor types. However, the role of exosomal circRNAs in bacterial infection remains elusive.MethodsWe extracted and identified exosomes from the culture medium of PIEC cells infected with or without Glaesserella parasuis. RNA sequencing analysis was performed on the exosomes to screen and identify circRNAs (circHIF1α) associated with Glaesserella parasuis infection. PIEC cells were infected with Staphylococcus aureus or Streptococcus suis 2 to further determine whether exosome-derived circHIF1α was the crucial circHIF1α associated with bacterial infections. The transmission process of exosomes and their circHIF1α between cells was clarified via exosome tracing and co-culture assay. Moreover, the mechanism of circHIF1α being packaged into exosomes was explored, and the effects of exosomes and their circHIF1α on cell proliferation, DNA damage and cell cycle were analyzed. In addition, the binding mode and site of interacting proteins with circHIF1α were further determined. In vivo and in vitro, the role of exosomes and their circHIF1α in host resistance to bacterial infection was confirmed.ResultsWe first discovered a new circHIF1α that was very stable and detectable, encapsulated into exosomes by hnRNPA2B1, and whose expression in exosomes of bacterially infected PIEC cells significantly decreased. Additionally, exosomal circHIF1α reduced bacterial infection both in vitro and in vivo and suppressed the growth of reception cells. Mechanistically, the circHIF1α interacted with the KH domain of IGF2BP3 in an m6A-modified manner, which mediated DNA damage to arrest the cells at the G1/S phase through the interaction between the regulator of Chromosome Condensation 2 (RCC2) and γ-H2AX protein. Exosomal circHIF1α is a unique therapeutic target for bacterial infection since this work highlights its critical function in fighting bacterial infection.

Prolonged Survival of Neutrophils Induced by Tumor-Derived G-CSF/GM-CSF Promotes Immunosuppression and Progression in Laryngeal Squamous Cell Carcinoma.

Tumor-associated neutrophils (TANs) play a crucial role in tumor progression and exhibit prolonged survival. However, the mechanism underlying their extended lifespan and significance in laryngeal squamous cell carcinoma (LSCC) remains unclear. Herein, it is observed that apoptosis of TANs is significantly delayed owing to induction by tumor-derived G-CSF and GM-CSF through the activation of the PI3K-AKT signaling pathway, upregulation of anti-apoptotic Mcl-1 expression, and downregulation of activated Caspase-3 levels. It is found that prolonged survival of TANs leads to the accumulation of aged CXCR4+ neutrophils that exhibit potent immunosuppressive properties and are associated with poor patient prognosis. Furthermore, extended survival promotes the enhanced immunosuppressive function of CD8+ T cells by TANs, thereby facilitating the in vitro and in vivo progression and growth of human LSCC tumors. Importantly, this effect could be reversed by blocking G-CSF and GM-CSF stimulation of neutrophils. These findings elucidate the pivotal role of pathologically prolonged neutrophil survival in impairing CD8+ T cell immunity and suggest targeting it as a potential therapeutic strategy for tumors.

Tumor stage-driven disruption of NK cell maturation in human and murine tumors

Tumor stage-driven disruption of NK cell maturation in human and murine tumors

Low SMARCD3 expression is associated with poor prognosis in patients with prostate cancer.

SWI/SNF complexes represent a family of multi-subunit chromatin remodelers that are affected by alterations in >20% of human tumors. While mutations of SWI/SNF genes are relatively uncommon in prostate cancer (PCa), the literature suggests that deregulation of various subunits plays a role in prostate tumorigenesis. To assess SWI/SNF functions in a clinical context, we studied the mutually exclusive, paralogue accessory subunits SMARCD1, SMARCD2, and SMARCD3 that are included in every known complex and are sought to confer specificity. Performing immunohistochemistry (IHC), the protein levels of the SMARCD family members were measured using a tissue microarray (TMA) comprising malignant samples and matching healthy tissue of non-metastatic PCa patients (n = 168). Moreover, IHC was performed in castration-resistant tumors (n = 9) and lymph node metastases (n = 22). To assess their potential role as molecular biomarkers, SMARCD1 and SMARCD3 protein levels were correlated with clinical parameters such as T stage, Gleason score, biochemical recurrence, and progression-free survival. SMARCD1 protein levels in non-metastatic primary tumors, lymph node metastases, and castration-resistant samples were significantly higher than in benign tissues. Likewise, SMARCD3 protein expression was elevated in tumor tissue and especially lymph node metastases compared to benign samples. While SMARCD1 levels in primary tumors did not exhibit significant associations with any of the tested clinical parameters, SMARCD3 exhibited an inverse correlation with pre-operative PSA levels. Moreover, low SMARCD3 expression was associated with progression to metastasis. In congruence with previous literature, our results implicate that both SMARCD1 and SMARCD3 may exhibit relevant functions in the context of prostate tumorigenesis. Moreover, our approach suggests a potential role of SMARCD3 as a novel prognostic marker in clinically non-metastatic PCa.

Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions.

Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity.

Distant Metastases of Breast Cancer Resemble Primary Tumors in Cancer Cell Composition but Differ in Immune Cell Phenotypes.

Breast cancer is the most commonly diagnosed cancer in women, with distant metastasis being the main cause of breast cancer-related deaths. Elucidating the changes in the tumor and immune ecosystems that are associated with metastatic disease is essential to improve understanding and ultimately treatment of metastasis. Here, we developed an in-depth, spatially resolved single-cell atlas of the phenotypic diversity of tumor and immune cells in primary human breast tumors and matched distant metastases, using imaging mass cytometry to analyze a total of 75 unique antibody targets. While the same tumor cell phenotypes were typically present in primary tumors and metastatic sites, suggesting a strong founder effect of the primary tumor, their proportions varied between matched samples. Notably, the metastatic site did not influence tumor phenotype composition, except for the brain. Metastatic sites exhibited a lower number of immune cells overall, but had a higher proportion of myeloid cells as well as exhausted and cytotoxic T cells. Myeloid cells showed distinct tissue-specific compositional signatures and increased presence of potentially matrix remodeling phenotypes in metastatic sites. This analysis of tumor and immune cell phenotypic composition of metastatic breast cancer highlights the heterogeneity of the disease within patients and across distant metastatic sites, indicating myeloid cells as the predominant immune modulators that could potentially be targeted at these sites.

Development of tripeptide-cyclotriphosphazene derivatives: In vitro cytotoxicity, genotoxicity studies and molecular docking analysis within ovarian and prostate cancer cell line receptors

Peptide-phosphazene compounds are important compounds of growing interest in biomedical research and have potential therapeutic effects. The tripeptide-cyclotriphosphazenes conjugates were synthesized and analyzed for their molecular docking analysis, visualizing their binding profiles within various cancer cell line receptors and tested in vitro cytotoxic and genotoxic properties. Determining in vitro cytotoxic studies of obtained compounds displayed cytotoxic effect against two selected human cancer cell lines, including ovarian (A2780) and prostate (PC-3), cancer cells. The compound DTAP demonstrated significantly higher efficacy at 100 µM in the PC-3 cancer cell line compared to the reference drug docetaxel at 50 µM. Among the tripeptide-phosphazene conjugtates, DTGG demonstrates the most promising anticancer activity with a logIC50 of 1.23 µM, forming five hydrogen bonds and a favorable salt bridge interaction, along with several hydrophobic interactions, thereby stabilizing its binding within the human ovarian tumor domain based on molecular docking analysis. The derivative DTGP emerges as the most potent among the DTG derivatives, achieving a ΔG model value of −108 kcal/mol, primarily due to a π-cationic interaction with the LYS204 amino acid in chain C, which significantly enhances its binding affinity. Additionally, DNA damage studies on human ovarian and prostate cancer cell lines determined that cell death due to DNA damage was the basis of the decrease in cell viability. These results support the evaluation of the compounds as potential drug candidates.

Sialylated glycoproteins suppress immune cell killing by binding to Siglec-7 and Siglec-9 in prostate cancer.

Prostate cancer is the second leading cause of male cancer death in the U.S. Current immune checkpoint inhibitor-based immunotherapies have improved survival for many malignancies; however, they have failed to prolong survival for prostate cancer. Siglecs (sialic acid-binding immunoglobulin-like lectins) are expressed on immune cells and regulate immune responses and function. Siglec-7 and Siglec-9 contribute to immune evasion by interacting with their ligands. However, the role of Siglec-7/9 receptors and their ligands in prostate cancer remains poorly understood. Here, we find that Siglec-7 and Siglec-9 are associated with poor prognosis in prostate cancer patients, and are highly expressed in myeloid cells, including macrophages, in prostate tumor tissues. Siglecs-7 and -9 ligands were expressed in prostate cancer cells and human prostate tumor tissues. Blocking the interactions between Siglec-7/9 and sialic acids inhibited prostate cancer xenograft growth and increased immune cell infiltration in humanized mice in vivo. Using a CRISPRi screen and mass spectrometry, we identified CD59 as a candidate Siglec-9 ligand in prostate cancer. The identification of Siglecs-7 and -9 as potential therapeutic targets, including CD59/Siglec-9 axis, opens up opportunities for immune-based interventions in prostate cancer.

Gasdermin D promotes development of intestinal tumors through regulating IL-1β release and gut microbiota composition

The interplay between gut microbiota and host is crucial for maintaining host health. When this balance is broken, various diseases can arise, including colorectal cancer (CRC). However, the mechanism by which gut microbiota and host interactions mediate CRC development remains unclear. Here, we found that Gasdermin D (GSDMD), an inflammasome effector responsible for forming membrane pores to mediate cell pyroptosis, was upregulated in both human and mouse intestinal tumor samples. GSDMD deficiency significantly suppressed intestinal tumor development in Apcmin/+ mice, a spontaneous CRC mouse model. Apcmin/+Gsdmd−/− mice exhibited reduced IL-1β release in the intestine, and the administration of recombinant mouse IL-1β partially restored intestinal tumor development in Apcmin/+Gsdmd−/− mice. Moreover, 16s rRNA sequencing showed a substantial increase in Lactobacillus abundance in the feces of Apcmin/+Gsdmd−/− mice compared to Apcmin/+ mice. Concurrently, Kynurenine (Kyn), a metabolite derived from host tryptophan (Trp) metabolism, was significantly decreased in the feces of Apcmin/+Gsdmd−/− mice, as shown by metabolite analysis. Additionally, Kyn levels were inversely correlated with Lactobacillus abundance. Furthermore, the administration of exogenous Kyn also promoted intestinal tumor development in Apcmin/+Gsdmd−/− mice. Thus, GSDMD promotes spontaneous CRC development through increasing IL-1β release and Kyn production. Our data suggest an association between GSDMD, gut microbiota, the host Trp/Kyn pathway, and CRC development.

Chronic exposure to hexavalent chromium induces esophageal tumorigenesis via activating the Notch signaling pathway.

Hexavalent chromium Cr(VI), as a well-established carcinogen, contributes to tumorigenesis for many human cancers, especially respiratory and digestive tumors. However, the potential function and relevant mechanism of Cr(VI) on the initiation of esophageal carcinogenesis are largely unknown. Here, immortalized human esophageal epithelial cells (HEECs) were induced to be malignantly transformed cells, termed HEEC-Cr(VI) cells, via chronic exposure to Cr(VI), which simulates the progress of esophageal tumorigenesis. In vitro and in vivo experiments demonstrated that HEEC-Cr(VI) cells obtain the ability of anchorage-independent growth, greater proliferative capacity, cancer stem cell properties, and the capacity to form subcutaneous xenografts in BALB/c nude mice when compared to their parental cells, HEECs. Additionally, HEEC-Cr(VI) cells exhibited weakened cell motility and enhanced cell adhesion. Interestingly, HEECs with acute exposure to Cr(VI) failed to display those malignant phenotypes of HEEC-Cr(VI) cells, suggesting that Cr(VI)‍-induced malignant transformation, but not Cr(VI) itself, is the cause for the tumor characteristics of HEEC-Cr(VI) cells. Mechanistically, chronic exposure to Cr(VI) induced abnormal activation of Notch signaling, which is crucial to maintaining the capacity for malignant proliferation and stemness of HEEC-Cr(VI) cells. As expected, N-‍[N-‍(3,5-difluorophenacetyl)‍-L-alanyl]‍-S-phenylglycine t-butyl ester (DAPT), an inhibitor for the Notch pathway, drastically attenuated cancerous phenotypes of HEEC-Cr(VI) cells. In conclusion, our study clarified the molecular mechanism underlying Cr(VI)‍-induced esophageal tumorigenesis, which provides novel insights for further basic research and clinical therapeutic strategies about Cr(VI)‍-associated esophageal cancer.

Establishing Patient-Derived Xenograft (PDX) Models of Lymphomas.

Patient-derived xenograft (PDX) models of lymphoma typically involve the injection of human tumor cells into an immunocompromised murine host. PDXs have the advantage that the tumor cells grow in a 3D environment within the mouse, meaning the selection pressure of in vitro establishment is avoided and the tumor cells better maintain their genetic heterogeneity. Here, we outline a method for producing and maintaining a PDX model of lymphoma. We describe three different methods to isolate a single cell suspension of the primary patient tumor, followed by either subcutaneous or intraperitoneal injection into an immunocompromised mouse. We then detail how to monitor tumor growth and how to harvest, passage, and store the tumors once they have grown. We highlight and discuss important protocol considerations including technical hints as well as the advantages and disadvantages of the methods described.

Conditional Pten inactivation in pituitary results in sex-specific prolactinoma formation

Pituitary tumors, including prolactinomas, present significant clinical challenges that require a deeper understanding of their molecular roots for improved diagnostics and therapies. Here, we investigate the role of the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K) pathway in pituitary tumorigenesis using a mouse model. Conditional knockout of Pten in all pituitary cell lineages resulted in prolactinoma formation exclusively in female mice, demonstrating the critical role of PTEN in pituitary homeostasis. While Pten inactivation induced Akt activation in all pituitary cells, only prolactin-producing cells exhibited tumorigenic changes, suggesting specific cell-type effects. Histological and molecular analyses of prolactinomas revealed similarities with human pituitary tumors, such as decreased vascularization and cell adhesion proteins and increased accumulation of cell cycle proteins. Notably, prolactinomas displayed diminished levels of phosphorylated extracellular signal-regulated kinase (ERK), implicating downregulation of ERK in tumorigenesis. Finally, we analyzed PTEN/PI3K activation in a collection of human pituitary tumors. Overall, our study delineates the intricate interplay between the PTEN and ERK signaling pathways, providing insights into sex-specific mechanisms of pituitary tumorigenesis and potential therapeutic strategies for prolactinomas.

Abstract A031: Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy

Abstract Preclinical and clinical studies have used antibodies to deplete CD4-expressing cells to promote anti-tumor immunity. Specifically, a monoclonal anti-CD4 antibody (anti-CD4) has been employed to remove FOXP3+ CD4+ regulatory T cells (Tregs), which are known to impede anti-tumor immunity. Our previous studies demonstrated that administering anti-CD4 during B16 melanoma tumor growth in mice leads to the generation of tumor Ag-specific CD8 T cells that disseminate and form memory following surgical tumor excision. However, the importance of co-depleting other CD4-expressing cell compartments for anti-CD4 treatment efficacy are unknown. We utilized flow cytometry and single-cell RNA sequencing (scRNAseq) to evaluate the primary response of tumor Ag-specific CD8 T cells (pmel) in B16 melanoma tumor bearing mice treated with anti-CD4. Responses were compared to those induced by anti-PD1 and anti-CTLA4 dual immune checkpoint blockade (ICB). To determine the importance of homeostatic space in priming, homeostatic expansion and priming were decoupled, and neutralizing antibodies were administered. Treg-depleted (Foxp3-DTR) mice were used for comparison, and Batf3-/- mice and IL-2R blocking antibodies were used to determine requirements for CD8 T cell priming. Finally, scRNAseq was conducted on CD4 T cells from human melanoma tumors previously treated with ICB to characterize transcriptional properties of intratumoral CD4 T cells. Compared to ICB, anti-CD4 treatment elicited a more robust, disseminated, and persistent primary tumor Ag-specific CD8 T cell response. ScRNAseq revealed that anti-CD4 treatment induced more stem-like (TCF1+) memory precursor cells, whereas ICB induced more differentiated effector (Gzmb+) cells. Removal of homeostatic space or blockade of IL-7R and IL-15 during priming did not diminish pmel responses in anti-CD4 treated mice, indicating that homeostatic space was not required. Importantly, pmel cells were shown to produce their own IL-2, and pmel priming was significantly lower after IL-2 receptor blockade, suggesting dependence on autocrine IL-2 signals. Using DT-treated FOXP3-DTR mice, we found that targeted Treg depletion was insufficient for the pmel priming that disseminated and formed memory, indicating that total CD4 T cell depletion was required for this systemic response. Depletion of total CD4 T cells uniquely increased cDC1 proportions in tumor-draining LNs as well as their expression of CD86. However, treatment of tumor-bearing RAG-/- mice with anti-CD4 confirmed that depletion of CD4-expressing APC compartments does not contribute to anti-CD4 treatment efficacy. ScRNAseq of CD4 T cells from human melanoma tumors showed dominance of Tr1-like cells and other subsets without transcriptional evidence of helper differentiation, despite prior ICB. In mice, combination of anti-CD4 with dual ICB restored the systemic response of tumor-specific CD8 T cells. This study underscores the importance of eliminating conventional CD4 T cells in tumor-bearing hosts to elicit systemic primary and memory CD8 T cell responses. Citation Format: Christo P. C. Dragnev, Delaney E. Ramirez, Tyler G. Searles, Nathaniel Spicer, TIffany Chen, J. Louise Lines, Aaron R. Hawkes, Wilson L. Davis III, Asmaa Mohamed, Keisuke Shirai, Joesph D. Phillips, Pamela C. Rosato, Yina H. Huang, Mary Jo Turk. Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A031.

Roles of Chemokine Axes in Breast Cancer.

Chemokines bind to specific chemokine receptors, known as cell surface G protein-coupled receptors, constructing chemokine axes which lead to cell migration and invasion in developmental stage, pathophysiological process, and immune reactions. The chemokine axes in the tumor microenvironment are involved in tumor growth, angiogenesis, cancer stem-like cell properties, metastasis, and chemoresistance, modifying tumor immune contexture and cancer progression. Clinical features, including tumor state, grade, lymph node metastasis, and cancer subtypes, are related to the specific chemokine axes, which play a significant role in immune contexture and cell to cell interaction in the tumor microenvironment, followed by altered cancer prognosis and overall survival. The present review summarizes the role of chemokine axes in breast cancer, based on data obtained from cell line and animal models and human tumor samples. This review provides information that understand the important roles of each chemokine axis in breast cancer, probably offering a clue of adjuvant therapeutic options to improve the quality of life and survival for patients with breast cancer.

PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

P21 activated kinase 6 (PAK6) is a serine-threonine kinase with physiological expression enriched in the brain and overexpressed in a number of human tumors. While the role of PAK6 in cancer cells has been extensively investigated, the physiological function of the kinase in the context of brain cells is poorly understood. Our previous work uncovered a link between PAK6 and the Parkinson’s disease (PD)-associated kinase LRRK2, with PAK6 controlling LRRK2 activity and subcellular localization via phosphorylation of 14–3–3 proteins. Here, to gain more insights into PAK6 physiological function, we performed protein-protein interaction arrays and identified a subgroup of PAK6 binders related to ciliogenesis. We confirmed that endogenous PAK6 localizes at both the centrosome and the cilium, and positively regulates ciliogenesis not only in tumor cells but also in neurons and astrocytes. Notably, PAK6 rescues ciliogenesis and centrosomal cohesion defects associated with the G2019S but not the R1441C LRRK2 PD mutation. Since PAK6 binds LRRK2 via its GTPase/Roc-COR domain and the R1441C mutation is located in the Roc domain, we used microscale thermophoresis and AlphaFold2-based computational analysis to demonstrate that PD mutations in LRRK2 affecting the Roc-COR structure substantially decrease PAK6 affinity, providing a rationale for the differential protective effect of PAK6 toward the distinct forms of mutant LRRK2. Altogether, our study discloses a novel role of PAK6 in ciliogenesis and points to PAK6 as the first LRRK2 modifier with PD mutation-specificity.

Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models.

Despite the cumulative evidence supporting HER3 as a target for antitumor therapies, no agents targeting HER3 have been approved for cancer treatment. Most of the agents evaluated in preclinical and clinical trials have been specific monoclonal antibodies (MAbs), with few examples of active immunotherapy directed against this receptor. However, some cancer vaccine formats may generate polyclonal antibodies (PAbs) that replicate the diverse effector mechanisms of MAbs, including ligand neutralization and receptor degradation. In this study, we developed a protein subunit-based monovalent vaccine candidate targeting the extracellular domain (ECD) of HER3. Immunization of mice with a formulation targeting murine ErbB3-ECD successfully overcome tolerance to this self-antigen, inducing high titers of ErbB3-specific PAbs. The antitumor potential of this formulation and the induced PAbs was demonstrated in vivo and in vitro in an ErbB3-overexpressing 3LL-D122-derived tumor model. The immunogenicity of the HER3-ECD-based vaccine candidate was confirmed by the induction of high titers of HER3-specific PAbs. Consistent with the initial results, HER3-ECD-targeting PAbs were cytotoxic in several human epithelial tumor cell lines and exerted antitumor effects in vivo. These results support the value of HER3 as a tumor antigen and the effector mechanisms of HER3-specific therapeutic MAbs, while suggesting the potential of the proposed vaccine candidate for the treatment of HER3-expressing carcinomas.

TRIP13 is Critical for the Survival of B-cell Lymphomas and Myeloid Leukemias In Vitro

Hematologic malignancies represent a diverse group of blood tumors accounting for approximately 10% of all cancer cases in the US. Based on the site of manifestation, presumed cell of origin, genetic abnormalities, and clinical features, we recognize more than 100 clinically important subtypes of hematologic malignancies broadly categorized into three main groups: leukemia, lymphoma, and multiple myeloma. While the prognosis for patients with specific subtypes of hematologic malignancies, such as Hodgkin lymphomas or chronic lymphocytic leukemia has significantly improved over the last several years, other subtypes such as acute myeloid leukemia or non-Hodgkin lymphomas still present with significant treatment challenges. A better understanding of molecular drivers of tumor maintenance is needed to improve existing therapies. Thyroid hormone receptor interactor 13 (TRIP13) is an AAA + ATPase that plays an important yet poorly understood role in the regulation of the mammalian cell cycle. The TRIP13 gene is highly expressed in various human tumors including colorectal carcinoma and glioblastoma, and it seems to promote tumorigenesis. Recently, we identified TRIP13 as a gene overexpressed in Peripheral T-cell lymphomas where it plays a critical role in tumor maintenance by regulating the G2-M phase of the cell cycle. However, the biological activities of TRIP13 in non-T cell hematologic malignancies have not been investigated to date. Here we used shRNA-mediated knockdown and a small molecule inhibitor to downregulate TRIP13 in B-cell lymphoid and myeloid lineages. We found genetic downregulation inhibited the proliferation of both B-cell lymphomas and acute myeloid leukemia cell lines in vitro. The pharmacologic inhibition of TRIP13 effectively induced the G0/G1 cell cycle arrest and apoptosis of B-cell lymphomas. Altogether these data demonstrate that TRIP13 is critical for the maintenance of hematologic malignancies of B-cell and myeloid malignancies in vitro. Thus, TRIP13 might be a good target in the treatment of various types of hematologic malignancies.

Evaluating glycocalyx morphology and composition in frozen and formalin-fixed liver tumor sections

BackgroundThe glycocalyx (GCX) is a glycan structure on the vascular endothelium and cancer cells. It is crucial for blood flow regulation, tumor invasion, and cancer drug resistance. Understanding the role of GCX in human tumors could help develop new cancer biomarkers and therapies. AimThis study aimed to demonstrate microstructural changes in human primary and metastatic liver tumors (henceforth termed liver tumors) by visualizing GCX using surgical specimens and comparing formalin-fixed paraffin-embedded sections (FFPEs) with frozen sections. The results of lectin staining were also compared between frozen and FFPE specimens to determine which was more useful for accurately assessing GCX structure and composition. MethodsLiver tumors and normal tissue samples from three patients were collected and processed into FFPEs and frozen sections, respectively. Lanthanum nitrate staining and scanning electron microscopy (SEM) were used to assess the GCX structures. Twenty lectins were analyzed for their glycan components in the samples. ResultsSEM revealed significant differences in GCX morphology among the cancer specimens. Frozen sections provided a more accurate GCX evaluation than FFPEs, showing distinct glycan compositions in hepatocellular carcinoma, colorectal carcinoma liver metastases, and melanoma liver metastases. Hepatocellular carcinoma samples exhibited a loss of N-acetylgalactosamine-related lectins. ConclusionThe results revealed that liver tumors have distinct and bulky GCX compared to normal liver tissue, while frozen sections are more reliable for GCX evaluation. These findings highlight glycan alterations in liver tumors and contribute to the development of new cancer therapies targeting GCX on tumor cell surfaces.

Effects of Thyroid Hormones on Cellular Development in Human Ovarian Granulosa Tumor Cells (KGN).

Ovarian cancer is a common malignant tumor in the female reproductive system, and Granulosa cell tumor (GCT) of the ovary is a rare type of ovarian cancer, which significantly threatens women's reproductive health. It has been reported that dysregulation of thyroid hormones (THs) may be closely related to the progression and prognosis of ovarian cancer. Moreover, THs regulate phosphorylation of signal transducer and activator of transcription (STAT3) and Octamer-binding transcription factor 4 (OCT4) expression. It has been reported that STAT3 and OCT4 play important roles in cellular development and tumorigenesis. However, the mechanisms by which THs affect the development of GCT are still remained unclear. To evaluate the effect of THs on human ovarian granulosa tumor cells (KGN), cells were treated with 3,5,3' -triiodothyronine (T3). Oct4 small interfering (Oct4 siRNA) or STAT3 inhibitor C188-9 was also co-cultured with cells in some experiments, respectively. The cell viability, proliferation, and proteins content were detected by CCK-8, EdU, and Western Blotting, respectively. The results showed that T3 enhanced cell viability and proliferation. Moreover, T3 also increased the expression of thyroid hormone receptor (TR), p-STAT3, and OCT4 proteins. The effects of T3 on both p-STAT3 and OCT4 expression were blocked by TR antagonist 1-850. Meanwhile, C188-9, an inhibitor of STAT3, decreased T3-induced cellular viability, proliferation, and OCT4 expression, highlighting that p-STAT3 can regulate the expression of OCT4 and affect cellular viability, and proliferation. Furthermore, T3-induced cellular growth was reduced by Oct4 siRNA, which indicates that T3 regulates cellular development through OCT4. These findings suggest that T3 increases cellular development via OCT4, which is mediated by phosphorylation of STAT3, and TR is also involved in these processes.

CELL-INTRINSIC METABOLIC PHENOTYPES IDENTIfiED IN GLIOBLASTOMA PATIENTS USING MASS SPECTROMETRY IMAGING OF 13C-LABELED GLUCOSE METABOLISM

Abstract AIMS Glioblastoma (GB) is an inherently heterogenous and invasive primary brain tumour. Genetic and transcriptomic studies have attempted to classify GB into subtypes that can identify therapeutic vulnerabilities and predict survival. An outstanding question and technical challenge is to visualise the metabolism of a tumour within its native microenvironment and understand to what extent its metabolism is driven by the microenvironment. METHOD Patients with GB tumours were infused with [U-13C]glucose and intra-operative multi-regional tumour samples were rapidly snap frozen for metabolic tracing analysis using mass spectrometry imaging (MSI). Tissue microenvironment was assessed using multiplexing of antibodies on immunocytochemistry (IMC) of parallel sections. Isotope tracing in human derived neurospheres and patient derived xenografts were used to confirm the presence of metabolic phenotypes detected in the human samples and to test susceptibility of each phenotype to a panel of drugs targeting cellular metabolism. RESULTS Three metabolic signatures were identified in patient GB tumours: glycolytic, oxidative and a mixed glycolytic/oxidative phenotype. The phenotypes did not correlate with microenvironmental features, including proliferation rate, immune cell infiltration, and vascularisation. The phenotypes were retained when patient derived cells were grown in vitro, or as orthotopically implanted xenografts, and were robust to changes in oxygen concentration, demonstrating their cell intrinsic nature. The spatial extent of the regions occupied by cells displaying these distinct metabolic phenotypes are large enough to be detected using clinically applicable metabolic imaging techniques. The phenotypes also demonstrate differential drug sensitivities. CONCLUSION This is the first report to demonstrate high resolution imaging of metabolic fluxes in a human tumour in vivo. In conjunction with studies on patient-derived neurospheres and orthotopically implanted xenografts we have demonstrated the presence of different metabolic phenotypes within GB that are tumour cell intrinsic and largely independent of the tumour microenvironment. These can be imaged clinically and used to stratify patients for personalised therapy.