Marker Of Tumor Progression Research Articles

ExoPD-L1: an assistant for tumor progression and potential diagnostic marker.

The proliferation and function of immune cells are often inhibited by the binding of programmed cell-death ligand 1 (PD-L1) to programmed cell-death 1 (PD-1). So far, many studies have shown that this combination poses significant difficulties for cancer treatment. Fortunately, PD-L1/PD-1 blocking therapy has achieved satisfactory results. Exosomes are tiny extracellular vesicle particles with a diameter of 40~160 nm, formed by cells through endocytosis. The exosomes are a natural shelter for many molecules and an important medium for information transmission. The contents of exosomes are composed of DNA, RNA, proteins and lipids etc. They are crucial to antigen presentation, tumor invasion, cell differentiation and migration. In addition to being present on the surface of tumor cells or in soluble form, PD-L1 is carried into the extracellular environment by tumor derived exosomes (TEX). At this time, the exosomes serve as a medium for communication between tumor cells and other cells or tissues and organs. In this review, we will cover the immunosuppressive role of exosomal PD-L1 (ExoPD-L1), ExoPD-L1 regulatory factors and emerging approaches for quantifying and detecting ExoPD-L1. More importantly, we will discuss how targeted ExoPD-L1 and combination therapy can be used to treat cancer more effectively.

Tumor Lipid Signatures Are Descriptive of Acquisition of Therapy Resistance in an Endocrine-Related Breast Cancer Mouse Model.

The lipid metabolism adaptations of estrogen and progesterone receptor-positive breast cancer tumors from a mouse syngeneic model are investigated in relation to differences across the transition from hormone-dependent (HD) to hormone-independent (HI) tumor growth and the acquisition of endocrine therapy (ET) resistance (HIR tumors). Results are articulated with reported polar metabolome results to complete a metabolic picture of the above transitions and suggest markers of tumor progression and aggressiveness. Untargeted nuclear magnetic resonance metabolomics was used to analyze tumor and mammary tissue lipid extracts. Tumor progression (HD-HI-HIR) was accompanied by increased nonesterified cholesterol forms and phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens) and decreased relative contents of triglycerides and fatty acids. Predominating fatty acids became shorter and more saturated on average. These results were consistent with gradually more activated cholesterol synthesis, β-oxidation, and phospholipid biosynthesis to sustain tumor growth, as well as an increase in cholesterol (possibly oxysterol) forms. Particular compound levels and ratios were identified as potential endocrine tumor HD-HI-HIR progression markers, supporting new hypotheses to explain acquired ET resistance.

Acid sphingomyelinase expression is associated with survival in resectable pancreatic ductal adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is one of the most common cancers worldwide. Unfortunately, the prognosis of PDAC is rather poor, and for instance, in the USA, over 47,000 people die because of pancreatic cancer annually. Here, we demonstrate that high expression of acid sphingomyelinase in PDAC strongly correlates with long-term survival of patients, as revealed by the analysis of two independent data sources. The positive effects of acid sphingomyelinase expression on long-term survival of PDAC patients were independent of patient demographics as well as tumor grade, lymph node involvement, perineural invasion, tumor stage, lymphovascular invasion, and adjuvant therapy. We also demonstrate that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase promotes tumor growth in an orthotopic mouse model of PDAC. This is mirrored by a poorer pathologic response, as defined by the College of American Pathologists (CAP) score for pancreatic cancer, to neoadjuvant therapy of patients co-treated with functional inhibitors of the acid sphingomyelinase, in particular tricyclic antidepressants and selective serotonin reuptake inhibitors, in a retrospective analysis. Our data indicate expression of the acid sphingomyelinase in PDAC as a prognostic marker for tumor progression. They further suggest that the use of functional inhibitors of the acid sphingomyelinase, at least of tricyclic antidepressants and selective serotonin reuptake inhibitors in patients with PDAC, is contra-indicated. Finally, our data also suggest a potential novel treatment of PDAC patients with recombinant acid sphingomyelinase.Key messagesPancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis.Expression of acid sphingomyelinase (ASM) determines outcome of PDAC.Genetic deficiency or pharmacologic inhibition of ASM promotes tumor growth in a mouse model.Inhibition of ASM during neoadjuvant treatment for PDAC correlates with worse pathology.ASM expression is a prognostic marker and potential target in PDAC.

Hemostasis parameters as prognostic biomarkers in gynecological cancer patients

Hypercoagulation is a typical condition for cancer patients. In addition, various arms of the hemostasis system become involved in tumor growth, invasion, metastasis, neoangiogenesis as well as immunoevasion. The magnitude of activated hemostasis is driven by tumor phenotype. Parameters characterizing coagulation, intensity of fibrinolysis processes, platelet aggregation and activation as well as endothelial activation mirror magnitude of hemostasis activation in oncological process, but may also be considered as candidate markers of tumor progression and predictors of oncological diseases outcome. This review summarizes the study data assessing a value of predictive hemostasis biomarkers for overall survival and response to therapy in oncogynecology and mammology. The most promising biomarkers have been identified and used in the future for risk stratification as well as personalized patient management.

Visualizing Galectin-3 Binding Protein Expression with ImmunoPET.

Galectin-3 binding protein (Gal-3BP) is a glycoprotein that is overexpressed and secreted by several cancers and has been implicated as a marker of both tumor progression and poor prognosis in melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. The expression of Gal-3BP by a variety of neoplasms makes it an enticing target for both diagnostics and therapeutics, including immuno-positron emission tomography (immunoPET) probes and antibody-drug conjugates (ADCs). Herein, we report the development, in vitro characterization, and in vivo evaluation of a pair of Gal-3BP-targeting radioimmunoconjugates for 89Zr-immunoPET. A humanized anti-Gal-3BP antibody, 1959, and its corresponding ADC, 1959-sss/DM4 (DM4 = ravtansine), were modified with desferrioxamine (DFO) to yield DFO-1959 and DFO-1959-sss/DM4 immunoconjugates bearing 1-2 DFO/monoclonal antibody. Both DFO-modified immunoconjugates retained their affinity for Gal-3BP in enzyme-linked immunosorbent assay experiments. The chelator-bearing antibodies were radiolabeled with zirconium-89 (t1/2 ≈ 3.3 d) to produce radioimmunoconjugates ─ [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 ─ with high specific activity (>444 MBq/mg, >12 mCi/mg) and stability (>80% intact after 168 h in human serum at 37 °C). In mice bearing subcutaneous Gal-3BP-secreting A375-MA1 xenografts, [89Zr]Zr-DFO-1959 clearly delineated tumor tissue, reaching a maximum tumoral activity concentration (54.8 ± 15.8%ID/g) and tumor-to-background contrast (tumor-to-blood = 8.0 ± 4.6) at 120 h post-injection. The administration of [89Zr]Zr-DFO-1959 to mice bearing subcutaneous Gal-3BP-expressing melanoma patient-derived xenografts produced similarly promising results. [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 exhibited nearly identical pharmacokinetic profiles in the mice bearing A375-MA1 tumors, though the latter produced higher uptake in the spleen and kidneys. Both [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 effectively visualized Gal-3BP-secreting tumors in murine models of melanoma. These results suggest that both probes could play a role in the clinical imaging of Gal-3BP-expressing malignancies, particularly as companion theranostics for the identification of patients likely to respond to Gal-3BP-targeted therapeutics such as 1959-sss/DM4.

Abstract LB233: Preclinical analysis identifies predictive biomarker and potential pathways of resistance to lurbinectedin treatment in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an exceptionally aggressive disease with limited treatment options that typically result in transient responses. SCLC is responsible for approximately 250,000 deaths globally per year. Major hurdles to improving SCLC treatment include the development of rapid chemo-resistance and limited second-line therapies. Lurbinectedin is FDA approved as a second-line treatment for SCLC but shows a response in a subset of patients. Therefore, improved mechanistic understanding and identifying predictive biomarkers of lurbinectedin treatment is a major unmet clinical need. Methods: We treated SCLC cell lines and patient-derived xenograft (PDX) models from all major SCLC subtypes with lurbinectedin. Lurbinectedin-mediated changes in signaling pathways were studied by bulk RNA sequencing, western blot, and flow cytometry. Anti-tumor efficacy and toxicity studies were performed in vivo. Result: All human and PDX-derived SCLC cell lines showed sensitivity to lurbinectedin at a nano-molar concentration ranging from 1.905 to 30 nM. Bulk RNA-seq analysis showed lurbinectedin induced changes in neuroendocrine phenotype, DNA damage response and tumor progression markers in vitro. Single agent treatment of lurbinectedin showed remarkable anti-tumor efficacy in an ASCL1-driven PDX model. RNA sequencing analysis identified modulation of genes in multiple signaling pathways including PI3K-AKT, apoptosis and EMT to be significantly associated with the lurbinectedin response in PDX models. Conclusion: There is an immediate need to understand the subsets of SCLC that would be most sensitive to lurbinectedin and identify predictive biomarkers. We demonstrate MYC as a predictive biomarker for lurbinectedin response. We are the first to show that single agent lurbinectedin shows remarkable anti-tumor efficacy in a ASCL1-driven PDX models of SCLC. Furthermore, our pre and post-lurbinectedin treatment transcriptomic analysis identify the pathways that may contribute to primary or acquired resistance to lurbinectedin in SCLC. Finally, we identity candidate targets that would guide the design of future combination clinical trials with lurbinectedin in SCLC. Citation Format: Subhamoy Chakraborty, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Preclinical analysis identifies predictive biomarker and potential pathways of resistance to lurbinectedin treatment in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB233.

SDHB immunohistochemistry for prognosis of pheochromocytoma and paraganglioma: A retrospective and prospective analysis.

Pheochromocytomas and paragangliomas (PCC/PGL) are rare neuroendocrine tumors and can secrete catecholamine. Previous studies have found that SDHB immunohistochemistry (IHC) can predict SDHB germline gene mutation, and SDHB mutation is closely associated with tumor progression and metastasis. This study aimed to clarify the potential effect of SDHB IHC as a predictive marker for tumor progression in PCC/PGL patients. We included PCC/PGL patients diagnosed in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from 2002 to 2014 for retrospective analysis and discovered that SDHB (-) staining patients had poorer prognoses. Then we examined SDHB protein expression by IHC on all tumors in the prospective series, which was composed of patients from 2015 to 2020 in our center. In the retrospective series, the median follow-up was 167 months, and during follow-up, 14.4% (38/264) patients developed metastasis or recurrence, and 8.0% (22/274) patients died. Retrospective analysis revealed that 66.7% (6/9) of participants in the SDHB (-) group and 15.7% (40/255) of those in the SDHB (+) group developed progressive tumors (OR: 10.75, 95% CI: 2.72-52.60, P=0.001), and SDHB (-) was independently associated with poor outcomes after adjusting by other clinicopathological parameters (OR: 11.68, 95% CI: 2.58-64.45, P=0.002). SDHB (-) patients had shorter disease-free survival (DFS) and overall survival (OS) (P<0.001) and SDHB (-) was significantly associated with shorter median DFS (HR: 6.89, 95% CI: 2.41-19.70, P<0.001) in multivariate cox proportional hazard analysis. In the prospective series, the median follow-up was 28 months, 4.7% (10/213) patients developed metastasis or recurrence, and 0.5% (1/217) patient died. For the prospective analysis, 18.8% (3/16) of participants in the SDHB (-) group had progressive tumors compared with 3.6% (7/197) in the SDHB (+) group (RR: 5.28, 95% CI: 1.51-18.47, P=0.009), statistical significance remained (RR: 3.35, 95% CI: 1.20-9.38, P=0.021) after adjusting for other clinicopathological factors. Our findings demonstrated patients with SDHB (-) tumors had a higher possibility of poor outcomes, and SDHB IHC can be regarded as an independent biomarker of prognosis in PCC/PGL.

Microbial Changes Associated With Oral Cavity Cancer Progression.

To examine the oral microbiome in the context of oral cavity squamous cell carcinoma. Basic science research. Academic medical center. Oral swabs were collected from patients presenting to the operating room for management of oral cavity squamous cell carcinoma and from age- and sex-matched control patients receiving surgery for unrelated benign conditions. 16S ribosomal RNA (rRNA) sequencing was performed on genetic material obtained from swabs. A bacterial rRNA gene library was created and sequence reads were sorted into taxonomic units. Thirty-one control patients (17 males) and 35 cancer patients (21 males) were enrolled. Ages ranged from 23 to 89 (median 63) for control patients and 35 to 86 (median 66) for cancer patients. Sixty-one percent of control patients and 63% of cancer patients were smokers. 16S analyses demonstrated a significant decrease in Streptococcus genera in oral cancer patients (34.11% vs 21.74% of the population, p = .04). Increases in Fusobacterium, Peptostreptococcus, Parvimonas, and Neisseria were also found. The abundance of these bacteria correlated with tumor T-stage. 16S rRNA sequencing demonstrated changes in bacterial populations in oral cavity cancer and its progression compared to noncancer controls. We found increases in bacteria genera that correspond with tumor stage-Fusobacteria, Peptostreptococcus, Parvimonas, Neisseria, and Treponema. These data suggest that oral cancer creates an environment to facilitate foreign bacterial growth, rather than implicating a specific bacterial species in carcinogenesis. These bacteria can be employed as a potential marker for tumor progression or interrogated to better characterize the tumor microenvironment.

Evaluation of total glutathione-s-transferase levels in serum of patients with oral malignancy.

Squamous cell carcinoma-related antigen is not always sensitive enough for the early detection of oral cancer which is why a new marker has been desired as a substitute to be applied for serum diagnosis of oral cancer. Reactive oxygen species (ROS) has been known to play an important role in carcinogenesis. Glutathione-s-transferases (GSTs) are a family of eukaryotic and prokaryotic phase-II metabolic isoenzymes involved in xenobiotic detoxification. This correlation of the ROS species function and their role in initiation and progression of cancer could be exploited as of diagnostic value. The biologic function of the GSTs in human head-and-neck squamous cell carcinomas has been studied by researchers at gross as well as molecular levels. Taking into consideration this scientific background, future scope and perspectives, we initiated this study. This study was performed as a prospective case-control in vitro analytical study with subjects (n = 40) fulfilling the prerequisite conditions and were compliant. The case group (n = 20) was subjects with histopathologically proven cases of oral malignancy and age- and sex-matched control group (n = 20). The enzyme GST was evaluated in sera of all participants and then comparison was done between two groups as well as correlation with histopathologic grading for oral malignancy. The mean serum GST activity in oral cancer patients was significantly higher than that of the control group. The present study has compared the alterations of enzyme in relation to histopathological grading of oral malignancy and found increased serum GST activity of well-differentiated and moderately differentiated carcinomas than the poorly differentiated carcinoma in terms of mean. Increased expression of the enzyme, as reported in the present study, can be due to tumor burden which attributes to overproduction of GST by cancer cells. The major clinical significance of the present study is that it gives important information regarding a new tumor progression and prognosis marker.

MicroRNA 200a as a histologically independent marker for meningioma recurrence: Results of a four microRNA panel analysis in meningiomas.

Meningiomas are mostly benign neoplasms of the central nervous system. Nevertheless there are recurrences in about 20% after surgical resection. Previous studies could reveal several predictors of meningioma recurrence. Tumor progression often is associated with a specific pattern of chromosome losses. Our study investigated the potential function of selected microRNAs as markers of tumor progression. By real-time polymerase chain reaction the expressions of microRNA 21-3p, 34a-3p, 200a-3p, and 409-3p were analyzed in solid tumor and in blood samples of 51 meningioma patients as well as in blood samples of 20 healthy individuals. Additionally, aberrations of parts of chromosomes 1, 14, 18, and 22 were analyzed by FISH. Tumor and blood samples were statistically analyzed, using Spearman's rank correlation coefficient as well as Mann-Whitney U- and Kruskal-Wallis-Test. MicroRNA 200a showed significantly lower expressions in recurrent meningiomas than in newly diagnosed ones. MicroRNA 409 in meningiomas was correlated significantly with tumor volume and showed a significant negative correlation with patient age. Significance was found between the expression patterns of microRNAs 34a and 200a with the respective aberrations of chromosome 1p and the microRNA 409 with aberration of chromosome 14. In the male cohort the expression of microRNA 200a in blood was significantly upregulated in patients compared to healthy volunteers. By our research the function of microRNA 200a was proved to detect meningioma patients by liquid biopsy. We detected microRNA 200a as a new biomarker to indicate meningioma recurrences. Future transferability to blood could be important for patient follow-up.

Aquaporin-4 Expression in Meningioma Malignancy Progression

Abstract Objectives The aim of the present study is to analyze if aquaporin-4 (AQP4) may also be a tumor progression marker for meningiomas. Methods This is an immunohistochemistry study realized at the Universidade de São Paulo, São Paulo, state of São Paulo, Brazil: frozen meningioma samples from 81 patients (57 females and 24 males, age range from 22 to 81 years old, average 56.5 ± 14.1 years old), including 57 meningiomas World Health Organization (WHO) grade I (GI); 19 grade II (GII), and 5 grade III (GIII) were analyzed. The relative expression level of AQP4 was analyzed by quantitative polymerase chain reaction (qPCR), using the SYBR Green approach and for staining detection. Tissue sections were routinely processed and subjected to antigen retrieval. Results The expression of AQP4 in meningioma samples ranged from 0 to 10.26, with a median of 0.001 in GI cases, of 0.008 in GII cases, and of 0.006 in GIII cases. Although not statistically significant (p = 0.942), GI meningiomas have a lower median AQP4 expression level than higher malignant grade cases. Conclusion The AQP4 gene and protein expressions presented no association with meningioma malignant progression.

PMON126 Null Cell Adenoma With Low Ki-67 Presenting as Recurrent Pituitary Mass

Abstract Background Pituitary adenomas account for 10% to 15% of all intracranial masses. Among these, approximately one-third are identified as nonfunctioning pituitary adenomas (NFPA) which have a heterogenous profile and an increased potential for relapse one to five years after pituitary surgery. In a retrospective analysis by Almeida et al,multiple surgical resections, elevated ki-67 and cavernous sinus invasion were predictive of recurrence. They typically present with symptoms of mass effect and most are macroadenomas at time of diagnosis. Clinical Case A 65-year-old female initially presented with bitemporal hemianopsia, galactorrhea and amenorrhea 28 years prior. She subsequently underwent transsphenoidal hypophysectomy with resolution of symptoms post operatively. She was clinically stable until 12 years after the surgery when she noted new onset peripheral visual field loss. Upon reevaluation, she was diagnosed with non-functioning pituitary macroadenoma for which she was advised gamma knife surgery but she opted a repeat transsphenoidal pituitary surgery. Her vision improved and she remained asymptomatic thereafter. Sixteen years after her second surgery, she developed blurring of vision. Cranial MRI was done which showed lobulated, heterogeneous hyperdense sellar-suprasellar mass measuring 55×36×47 mm extending to the bilateral sphenoid and ethmoid sinuses, left pterygopalatine fossa, left superior orbital fissure, probably the left foramen lacerum, and left cavernous sinus with encasement of the left internal carotid artery and associated erosion of the adjacent osseous structures. Visual field testing showed mild reduction in field sensitivity with consideration of media opacity and/or uncorrected error of refraction. Baseline hormonal work up was unremarkable. Hence, endoscopic endonasal transphenoidal, transethmoidal parasellar excision of sellar mass, with reconstruction via Hadad Flap was done. There were no intraoperative nor postoperative complications. The specimen was sent for histopathology with provisional anatomic diagnosis of pituitary adenoma. Immunohistochemistry stained negative for chromogranin and any of the pituitary hormones which was consistent with null cell adenoma. The Ki-67, an independent marker of tumor progression and recurrence, was low at less than 1%. However, after 1 month, repeat MRI showed  no significant change in the lobulated, heterogeneously enhancing mass centered in the sellar-suprasellar region. Thus, she received adjuvant radiotherapy with total dose of 5040cGy divided in 28 fractions and was advised close monitoring of pituitary MRI and/or development of any new symptoms. Conclusion In this case, although with low Ki-67, the presence of multiple surgeries and high Knosp grade were recognized as risk factors for its recurrence.Treatment of recurrent NFPA is multimodal which includes re-operation, radiosurgery and radiation therapy. A multidisciplinary team approach is required for its comprehensive management and long-term follow-up. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Loss of p16 expression is a risk factor for recurrence in sinonasal inverted papilloma.

The purpose of this study was to evaluate p16, p53, EGFR, pEGFR protein expression and HPV infection as possible markers of tumor progression in a series of sinonasal inverted papilloma (SNIP) and sinonasal squamous cell carcinoma (SNSCC). A series of 49 SNIP, 11 SNSCC associated with SNIP (SNIP-SNSCC) and 52 SNSCC not associated with SNIP were analyzed for p16, p53, EGFR, and phosphorylated EGFR (pEGFR) expression by immunohistochemistry. Human papillomavirus (HPV) infection status was evaluated by DNA-PCR. Results were correlated to clinical and follow-up data. Reduced or loss of p16 expression was observed in 18% SNIP, 64% SNIP-SNSCC and 87% of SNSCC. Reduced or loss p16 staining in SNIP correlated with shorter recurrent SNIP-free follow-up. In contrast, p16 expression was not predictive of recurrent SNSCC in cases with SNIP-SNSCC and SNSCC. P53, EGFR, and pEGFR expression did not differ between the tumor groups, nor were they related to recurrent SNIP-free follow-up or recurrent SNSCC. Oncogenic HPV types 16 and 18 were detected in 5% of SNIP and 18% of SNIP-SNSCC, but not in SNSCC. There was no correlation between HPV infection and &gt;70% p16 immunostaining. HPV infection appears to play a minor role in SNIP and SNSCC and p16 immunostaining does not appear a valid surrogate marker for HPV. However, reduced or loss p16 expression may have prognostic value as a risk marker for recurrent SNIP.

MicroRNA-570 targets the HSP chaperone network, increases proteotoxic stress and inhibits mammary tumor cell migration

The dynamic network of chaperone interactions known as the chaperome contributes significantly to the proteotoxic cell response and the malignant phenotype. To bypass the inherent redundancy in the network, we have used a microRNA (mir) approach to target multiple members of the chaperome simultaneously. We identified a potent microRNA, miR-570 that could bind the 3′untranslated regions of multiple HSP mRNAs and inhibit HSP synthesis. Transfection of cells with this miR species reduced expression of multiple HSPs, inhibited the heat shock response and reduced tumor cell growth while acted additively in combination with cytotoxic drugs. As overexpression of miR-570 elicited tumor suppressive effects, we inferred that this miR could play a potential role in inhibiting tumorigenesis and cancer cell growth. In accordance with this hypothesis, we determined a significant role for miR-570 in regulating markers of mammary tumor progression, including cell motility and invasion. Our data provide a proof of the principle that the tumor chaperome can be targeted by microRNAs suggesting a potential therapeutic avenue towards cancer therapy.

Chemically engineering the drug release rate of a PEG-paclitaxel conjugate using click and steric hindrance chemistries for optimal efficacy

A small molecule drug with poor aqueous solubility can be conjugated to a hydrophilic polymer like poly(ethylene glycol) (PEG) to form an amphiphilic polymer-drug conjugate that self-assembles to form nanoparticles (NPs) with improved solubility and enhanced efficacy. This strategy has been extensively applied to improve the delivery of several small molecule drugs. However, very few reports have succeeded to tune the rate of drug release from these NPs. To the best of our knowledge, there have been no reports of utilizing click and steric hindrance chemistry to modulate the drug release of self-assembling polymer-drug conjugates. In this study, we utilized click chemistry to conjugate methoxy-PEG (mPEG) to an anti-tumor drug, paclitaxel (PTX). A focused library of PTX-Rx-mPEG (x = 0, 1, 2) conjugates were synthesized with different chemical modalities next to the cleavable ester bond to study the effect of increasing steric hindrance on the self-assembly process and the physicochemical properties of the resulting PTX-NPs. PTX-R0-mPEG had no added steric hindrance (x = 0; minimal), PTX-R1-mPEG consisted of two methyl groups (x = 1: moderate), and PTX-R2-mPEG consisted of a phenyl group (x = 2: significant). Drug release studies showed that PTX-NPs released PTX at a decreased rate with increasing steric hindrance. Pharmacokinetic studies showed that the AUC of released PTX from the moderate-release PTX-R1-NP was approximately 20-, 6-, and 3-fold higher than that from free PTX, PTX-R0-NP and PTX-R2-NP, respectively. As a result, among these different PTX formulations, PTX-R1-NP showed superior efficacy in inducing tumor regression and prolonging the animal survival. The tumors treated with PTX-R1-NP displayed the lowest tumor progression markers (Ki68 and CD31) and the highest apoptotic marker (TUNEL) compared to the others. This work emphasizes the importance of taking a systematic approach in designing self-assembling polymer drug conjugates and highlights the potential of utilizing steric hindrance as a tool to tune the drug release rate from such systems.

A Comprehensive Evaluation of Prognostic Value and Immune Infiltration of KDM1 Family in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies in the world. Previous studies indicated that the expression of the KDM1 genes (KDM1s), members of the amine oxidase superfamily, has prognostic value for breast and prostate cancer and malignant neuroblastoma. This study aimed to investigate the expression of KDM1s, their prognostic value, and their correlation with immune infiltration in patients with HCC. Multiomics analyses were utilized to analyze differential expression, prognostic value, genetic alteration, and immune cell infiltration of KDM1s in patients with HCC. The high expression of KDM1A indicated poor overall survival (OS) and disease-free survival, whereas the high expression of KDM1B was significantly associated with poor OS. The genetic alterations and biological interaction network of KDM1s may provide detailed information for the dysregulated function of KDM1s in patients with HCC. KDM1-related signaling pathways and miRNA targets were explored and may provide value as therapeutic targets or tumor progression markers. The increased mRNA expression of KDM1s was significantly correlated with the infiltration of diverse immune cells in HCC. This data-driven study indicates that KDM1s are promising prognostic biomarkers for survival and have the potential to become novel molecular targets in HCC treatments.

Systematic Identification of the RNA-Binding Protein STAU2 as a Key Regulator of Pancreatic Adenocarcinoma

Simple SummaryPancreatic adenocarcinoma (PAAD) is one of the most common tumors of the gastrointestinal tract and is difficult to diagnose and treat due to tumor heterogeneity and the immunosuppressive tumor microenvironment. RNA-binding proteins have been studied and their dysregulation has been found to play a key role in altering RNA metabolism in various malignancies. STAU2 is one of them. To investigate the role of STAU2 in PAAD, we monitored the signaling pathway by regulating substrate mRNA and experimentally confirmed that STAU2 is the most potential biomarker for the occurrence and development of PAAD. Furthermore, we found that high expression of STAU2 not only contributes to immune evasion but also correlates with sensitivity to chemotherapeutic agents, suggesting that STAU2 may be a potential target for combined natural therapy. These results demonstrate that STAU2 is a novel prognostic and diagnostic biomarker for PAAD, revealing STAU2′s utility in cancer therapy and drug development.Pancreatic adenocarcinoma (PAAD) is a highly aggressive cancer. RNA-binding proteins (RBPs) regulate highly dynamic post-transcriptional processes and perform very important biological functions. Although over 1900 RBPs have been identified, most are considered markers of tumor progression, and further information on their general role in PAAD is not known. Here, we report a bioinformatics analysis that identified five hub RBPs and produced a high-value prognostic model based on The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets. Among these, the prognostic signature of the double-stranded RNA binding protein Staufen double-stranded RNA (STAU2) was identified. Firstly, we found that it is a highly expressed critical regulator of PAAD associated with poor clinical outcomes. Accordingly, the knockdown of STAU2 led to a profound decrease in PAAD cell growth, migration, and invasion and induced apoptosis of PAAD cells. Furthermore, through multiple omics analyses, we identified the key target genes of STAU2: Palladin cytoskeletal associated protein (PALLD), Heterogeneous nuclear ribonucleoprotein U (HNRNPU), SERPINE1 mRNA Binding Protein 1 (SERBP1), and DEAD-box polypeptide 3, X-Linked (DDX3X). Finally, we found that a high expression level of STAU2 not only helps PAAD evade the immune response but is also related to chemotherapy drug sensitivity, which implies that STAU2 could serve as a potential target for combinatorial therapy. These findings uncovered a novel role for STAU2 in PAAD aggression and resistance, suggesting that it probably represents a novel therapeutic and drug development target.

Proteomic alterations in extracellular vesicles induced by oncogenic PIK3CA mutations.

PIK3CA is one of the most frequently mutated genes in human cancers, with the two most prevalent activating mutations being E545K and H1047R. Although the altered intracellular signaling pathways in these cells have been described, the effect of these mutations on their extracellular vesicles (EVs) has not yet been reported. To study altered cellular physiology and intercellular communication through proteomic analysis of EVs, MCF10A cells and their isogenic mutant versions (PIK3CA E545K and H1047R) were cultured and their EVs enriched by differential ultracentrifugation. Proteins were extracted, digested with trypsin and the peptides labeled with tandem mass tag (TMT) reagents and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Four thousand six hundred and fifty-five peptides were identified from 579 proteins of which 522 proteins have been previously described in EVs. Relative quantitation revealed altered levels of EV proteins including several cell adhesion molecules. Mesothelin, E-cadherin, and epithelial cell adhesion molecule were elevated in both mutant cell-derived EVs. Markers of tumor invasion and progression like galectin-3 and transforming growth factor beta induced protein were increased in both mutants. Overall, activating mutations in PIK3CA result in altered EV composition with characteristic changes associated with these hotspot mutations.

Abstract 2025: Spatial transcriptomic profiling of progression markers in clear cell renal cell carcinoma

Abstract Next-generation sequencing enabled molecular landscaping and the discovery of novel tumor markers in ccRCC. However, the spatial relationships and histological features of the tumor microenvironment were lost during this process. Combining snRNA-seq, snATAC-seq, Spatial Transcriptomics (ST) technology, and immunofluorescence labeling, we discovered novel ccRCC progression tumor markers, and uncovered their spatial dependent expression pattern in both human ccRCC tumors and patient-derived xenograft (PDX). Using snRNA-seq and snATAC-seq, novel tumor progression markers such as NDRG1, MGST1, ABCC3 and PCSK6 were discovered, and their expressions were validated in ST. We found that one of the key novel tumor markers, CP, exhibited specific spatial expression patterns on tissue slides. The elevation of CP expression region is associated with a higher degree of hyalinization in ccRCC human tumor samples. Similarly, using immunofluorescence labeling, we validated the co-expression of CP and PCSK6 canonical ccRCC marker CA9. Overall, this study revealed novel ccRCC progression markers and linked their spatial expression pattern with histological features. Citation Format: Chia-Kuei Mo, Yige Wu, Terekhanova Nadezhda, Caravan Wagma, Preet Lal, Siqi Chen, Nataly Naser AL Deen, Ruiyang Liu, Yanyan Zhao, Kazuhito Sato, Lijun Yao, Mamatha Serasanambati, Andrew Shinkle, Reyka G. Jayasinghe, Li Ding, Feng Chen. Spatial transcriptomic profiling of progression markers in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2025.

Abstract LB025: Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation

Abstract Metastatic relapse is the major causes of mortality in patients with cancer and occur due to metastatic reactivation of dormant tumor cells. Early dissemination of tumor cells undergoing a protected period of dormancy in the target organs potentially explains this prevalent clinical behavior. Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases. Malat1 is one of the most abundant and highly conserved nuclear lncRNAs and have shown the associated with metastasis and serving as a predictive marker for various tumor progression. However, the correlation of tumor intrinsic lncRNAs in regulation of tumor dormancy and immune evasion are largely unknown. Using an in vivo screening platform for the isolation of genetic entities involved in either dormancy or reactivation of breast cancer tumor cells, we have identified Malat1 as a positive mediator of metastatic reactivation. To dissect the functional role of Malat1 in metastatic reactivation, we developed a clean Malat1 knockout (KO) model using paired gRNA CRISPR-Cas9 in metastatic murine syngeneic breast cancer. As a proof of concept, we also used inducible knockdown system under in vivo models. To delineate the immune microenvironment, we used single cell RNA-seq, ChIRP-seq, multicolor flowcytometry, RNA-FISH, and coculture experiments. Our data revealed that deletion of Malat1 induces dormancy and attenuated the metastatic colonization resulting in long-term survival of syngeneic mice model. In contrast, overexpression of Malat1 leads to metastatic reactivation of dormant breast cancer cells. Interestingly, 4T1-Malat1 KO dormant breast cancer cells exhibit metastatic outgrowth in T cells defective mice. Our single-cell RNA-seq and multicolor flowcytometry evaluation reveal enhanced T cells and reduced neutrophils proportions in mice with Malat1 KO cells. This indicates a critical role of immune microenvironment via Malat1-dependent immune evasion. Additionally, Malat1 KO inhibits cancer stemness properties. Similarly, RNA-seq and ChIRP-seq data suggest that KO of Malat1 hampers immune evasion and downregulates metastasis associated genes including Serpins and Wnts. Additionally, our data strongly suggests that Malat1 KO cells persists as non-proliferative dormant cells in lung due to CD8+ T cell-umpired immune activity. Our mechanistic studies showed that Malat1 regulated Sepinb6b blocks the CstG enzymatic activity and suppress cleavage of GsdmD and T cells induced pyroptosis. Interestingly, rescue experiments suggest that Malat1 or Serpinb6b protects T cell-induced cell death and induces dormancy re-awakening thereby rescue the metastatic potential of 4T1 Malat1 KO cells. Combination of Malat1 ASO with double immune checkpoint inhibitors greatly affects the metastatic outgrowth in breast cancer. Taken together, our studies demonstrate that tumor intrinsic Malat1 regulates Serpinb6b that suppresses CstG-GsdmD mediated pyroptosis which eventually promotes immune evasion and breast cancer dormancy metastatic reactivation. Citation Format: Dhiraj Kumar, Filippo Giancotti. Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB025.