Human Tumor Research Articles

Programmed-cell death ligand 1 (PD-L1) expression in equine sarcoids and squamous cell carcinoma.

Sarcoids and squamous cell carcinomas (SCCs) are the most concerning equine oncological diseases. Both tumors are challenging to manage due to their invasive behavior and high prevalence of recurrences. Furthermore, SCCs have a propensity to metastasize. Programed cell-death ligand 1 (PD-L1) has been one of the main therapeutic targets for immunotherapy in various human tumors. PD-L1 research in equine tumors is scarce and more efforts are necessary to understand the potential of this biomarker as a therapeutical target. Evaluate the immunohistochemical expression of PD-L1 in equine sarcoids and SCC. Thirteen equine tumors (seven sarcoids and 6 SCCs) were tested by immunohistochemistry and evaluated semi quantitatively to assess the percentage of positive cells. None of the sarcoids presented PD-L1 expression. Regarding SCC, 2 tumors presented <10% of labeled cells; 2 tumors presented 10%-25% of labeled cells and 2 tumors presented 25%-50% of labeled cells. There were statistically significant differences between sarcoids and SCC regarding the expression of PD-L1. Our results point to the fact that PD-L1 could be a potential therapeutic target against SCC, and also encourage in-depth studies in this area, with larger sample sizes.

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

The long noncoding RNA MANCR to promote and stabilize triple negative breast cancer.

e13150 Background: Cancer remains a leading cause of death worldwide, despite many advances in cancer treatment over the past decade. Triple Negative Breast Cancer (TNBC) is among the highest with a poor prognosis. Early diagnosis and modern treatment regimens for breast cancer (BC) have led to a high disease-free survival rate. Still, survival decreases significantly for patients with metastatic disease. Long non-coding RNAs are among a recent class of epigenetic regulators that function in the nucleus to support the stability of the cells and maintain the fidelity of chromatin interactions. Our laboratory discovered the long noncoding RNA MANCR (LINC00704) as being upregulated in human BC tumors and this is indicative of a worse survival rate than in patients with low MANCR-expressing tumors. MANCR is aberrantly expressed in TNBC cells, and these cells are highly dependent on MANCR to retain their tumorigenic characteristics. Methods: Our functional in vitro studies in MDA-MB-231 TNBC cells used GapmerRs to knockdown MANCR. For in vivo studies, TNBC cells were injected into the 4th mammary fat pad of mice, allowed initial tumor formation, and then treated the animals with 2 nmol/g of a negative control or MANCR targeting GapmeR. We also identified genome interaction sites at single nucleotide resolution by chromatin isolation by RNA purification (ChIRP)-seq to determine the mechanism of MANCR activity in TNBC cells. Results: We found that MANCR knockdown promotes DNA damage and decreases cell proliferation, migration, anchorage-independent colony formation, transwell invasion, and overall survival. Additionally, targeting MANCR in animals with existing tumors drastically inhibited tumor growth and the end-point tumor mass over time. The MANCR GapmeR treatment also inhibited the ability of TNBC cells to circulate and disseminate to distant organs. After performing ChIRP-seq in the MDA-MB-231 cells, we identified 988 genome-wide binding sites that exhibit MANCR interactions, of which 79% are intergenic and 21% are genic regions. Furthermore, 127 MANCR ChIRP peaks were found to overlap with fragile sites in the genome, indicating MANCR provides stability to these sites. Conclusions: These data suggest that targeting MANCR has therapeutic potential for patients with “MANCR-high” TNBC tumors by disrupting genome stability. Indeed, our in vivo studies demonstrate that “MANCR-high” TNBC tumors require MANCR to rapidly grow and promote disease progression. Lastly, many of the MANCR-chromatin interactions identified were found in intergenic regions and overlap with fragile sites within the genome. Collectively these data strongly indicate that MANCR stabilizes the TNBC genome, and disrupting genome stability by targeting MANCR has therapeutic potential.

Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs.

We aimed to assess the effects of a cocktail comprising three specific anti- HER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of each of the three antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phage-scFv cocktail/ kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20 mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Improved Mass Spectrometry–Based Methods Reveal Abundant Propionylation and Tissue-Specific Histone Propionylation Profiles

Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry–based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states.

The FIPO23 trial: First-in-human phase I/II study of XON7 in advanced solids tumors.

TPS2673 Background: XON7 is a first-in-class glyco-humanized polyclonal antibody (GH-pAb) targeting selectively multiple tumor-associated antigens. XON7 induces tumor cell apoptosis and promotes the elimination of tumor cells by immune effector cells through CDC (Complement Dependent Cytotoxicity) and ADCP (Antibody-Dependent Cell Phagocytosis). XON7 demonstrated anti-tumor efficacy in mice xenograft models with human solid tumors such as colon, prostate, lung and triple negative breast cancers. Safety pharmacology and toxicology studies in non-human primates demonstrated an acceptable safety profile for XON7. Taken together, these preclinical data support the initiation of human trials in patients with advanced solid tumors. Methods: First-in-patient, multicenter, open-label, two phases (dose-escalation (ESC) followed by dose expansion (EXP)) study of XON7 single agent, in patients with relapsed refractory, locally advanced or metastatic solid tumors without standard available treatments ( NCT06154291). Key eligibility criteria include disease progression after ≤ 4 lines of therapy; age &gt; 18 years; ECOG performance status ≤2; adequate organ functions; no known CNS involvement. All advanced or metastatic tumor types except glioblastoma, can be included during ESC. Primary endpoints: safety, tolerability and determination of MTD/RP2D. Secondary endpoints: pharmacokinetics (PK), pharmacodynamics, immunogenicity and preliminary anti-tumor activity (RECIST 1.1). AEs graded according to CTCAE 5.0. In the ESC part, XON7 is administered as 60-min intravenous infusion every 2 weeks according to an escalating schedule of doses from 1.5 to 20 mg/kg for up to 12, 28-day cycles. Dose escalation/de-escalation is based on the Bayesian Optimal Interval (BOIN) design, up to 45 pts are planned. At the recommended dose, up to 7 EXP cohorts (30 pts each) defined according to the primary tumor site are planned. Bayesian sequential monitoring will be used. Patient recruitment is planned or ongoing at 25 sites worldwide. Results: As of Feb 3rd, 2024, 3 pts are enrolled in the first dose cohort and have received XON7 at dose 1,5 mg/kg Q2W for the first 28-days cycle. Conclusions Accrual is ongoing in this first-in-human trial of XON7. Clinical trial information: NCT06154291 .

Safety and efficacy of B7-H3 targeting CAR-T cell therapy for patients with recurrent GBM.

2062 Background: Glioblastoma Multiforme (GBM) is one of the most deadly cancers, and nearly all patients experience recurrence upon the current standard of care for newly diagnosed GBM. The recurrent GBM (rGBM) exhibits a median survival of less than 8 months with limited therapeutic options. We have previously cloned the B7-H3 gene, characterized its function, demonstrated altered B7-H3 expression in human solid tumors, and identified B7-H3 as a promising therapeutic target. We present herein the preliminary findings of an investigator-initiated trial (NCT05241392), where patients with rGBM were treated with autologous chimeric antigen receptor-T (CAR-T) cells targeting B7-H3 (TX103). Methods: This is an open, single-arm, "3+3" dose-escalation and multiple-dose study. The primary objective was to evaluate the safety and the maximal-tolerated dose. Secondary objectives included survival outcomes, tumor responses, immunological responses, and pharmacokinetic parameters. Patients aged 18 to 75 were enrolled with confirmed recurrence by image scan and B7-H3 expression &gt;= 30% by immunohistochemistry analysis. TX103 was administered intracavity and/or intraventricularly to patients via an Ommaya reservoir. TX103 was given biweekly to patients each cycle at dose levels 1, 2, and 3 (20, 60, 150 million cells, respectively), with four cycles as one course. Results: Between March 2022 and January 2024, 13 patients received at least one intracranial infusion of TX103, with 3, 4, and 6 patients in dose levels 1, 2, and 3, respectively. The median number of infusion cycles was 4 (Min, Max 1,9). All patients were included in the safety analysis, revealing no dose-limiting toxicity or CAR-T treatment-related death. All patients experienced at least one adverse event (AE), and the treatment-related AEs (TRAEs) included cytokine release syndrome, increased intracranial pressure (ICP), headache, epilepsy, decreased level of consciousness, vomiting, and pyrexia. Most TRAEs were grade 1-2, while three grade-3 TRAEs were observed: one case of increased ICP in dose level 2, one case of epilepsy, and another case of decreased level of consciousness in dose level 3. As of January 2024, six patients from dose levels 1 and 2 were eligible for a 12-month survival assessment; the 12-month overall survival (OS) rate was 83.3% (95% CI: 58.3%~100%), and the median OS was 20.3 mo (95% CI: 20.3~not reach). Two of the three patients from dose level 2 achieved partial and complete responses, respectively. Following CAR-T administration, significant increases in cytokines, including IL-6 and IFN-γ, and elevated copy numbers of the CAR gene were observed in the cerebrospinal fluid, while only minimal elevations were noted in the peripheral blood. Conclusions: In this trial, treating rGBM with TX103 is deemed safe and tolerated. These interim results support further study using TX103 to enhance the survival outcomes in patients with rGBM.

Anticancer Properties of Different Varieties of Date Palm (Phoenix dactylifera L.) Leaf Extracts in Human Tumor Cells: a Comparative Study.

Plant polyphenols are nutraceutical components with relevant biological effects on human health. They act against development of several diseases including cancer. In this study, the methanolic extracts of four date palm Phoenix dactylifera leaves (Deglet Noor (DN), Barhee (B), Khalas (KS) and Khunezi (KZ)) collected from south Tunisia were preliminary analyzed for their effects against U87 (human glioblastoma) and MDA-MB-231 (human breast cancer) cell line development. Results showed that Barhee extract (30μg/mL) was the most efficient to reduce the growth of both tumor cells to about 40% (p < 0.05) without inducing cytotoxicity. Significantly, KS, KZ, DN and B extracts (30μg/mL) decreased MDA-MB-231 and U87 cell adhesion towards fibrinogen and fibronectin. Using integrin blocking antibodies, leaf extracts competitively decreased human glioblastoma cell attachment to immobilized antibodies by interfering to αvβ3 and α5β1 integrin receptors. At the same concentration, extracts decreased MDA-MB-23 and U87 cell migration performed with wound healing assay. Particularly, Barhee and Deglet Noor leaf extracts (30μg/mL) significantly reduced U87 cell invasion by 52.92% (p < 0.01) and 74.56% (p < 0.01), respectively. Collegially, our findings revealed beneficial proprieties of four varieties of date palm leaf especially those displayed by DN and B extracts that may serve as active candidates against human glioblastoma and breast cancer progression.

Feasibility study of a personalized tumor membrane vesicle vaccine immunotherapy for head and neck squamous cell carcinoma (HNSCC).

e14673 Background: Head and neck cancer exhibits a high degree of heterogeneity, with lack of common therapeutic targets. Our phase 1 clinical trial will evaluate a novel personalized immunotherapy approach using tumor membrane vesicles (TMV) prepared from the patients’ own surgically excised tumor tissue and modifying them by attaching GPI-B7-1 and GPI-IL-12 as immune boosting molecules. This personalized vaccine will then be administered to boost the tumor-specific immunity. We report here the feasibility of developing an effective personalized therapeutic cancer vaccine from surgically collected tumor tissue from patients with HNSCC. Methods: After obtaining informed consent, a total of 10 tumor specimens from 10 patients with HNSCC of various sites were collected (0.29-1.23 grams) and graded for cellularity by a pathologist. Tumor samples were then homogenized using sterile, disposable probes. The homogenate was ultracentrifuged over a sucrose gradient to enrich tumor membrane vesicles (TMV). Yield was evaluated by protein concentration and particle size was assessed by dynamic light scattering. TMV preparations were incorporated with GPI-B7-1 and GPI-IL-12 to generate the TMV vaccine using protein transfer, a spontaneous process. For the in vivo murine studies, TMV vaccine is prepared from murine oral cancer (MOC1) tumors and incorporated with murine GPI-B7-1 and GPI-IL-12. To investigate the protective anti-tumor immune response of TMV vaccine in combination with anti-PD1 antibody therapy, we used established murine oral cancer models. We administered two doses of TMV vaccine to naïve C57BL/6 mice and four doses of anti-PD-1 mAb. Mice were monitored for 21 months and then challenged with MOC1 tumor cells 1 week prior to euthanasia. Spleens were flash frozen in liquid nitrogen and stored in -80oC until RNA is isolated for RNA-seq analysis. Results: The yields of TMV were 2.5 – 5 mg per gram of tumor tissue from four human tumor samples. Further refinement of the TMV production process yielded up to 6 mg of TMV/gram of tumor tissue. All samples passed for incorporation of GPI-B7-1 and GPI-IL-12. GPI-B7-1 is able to bind to a natural ligand, CTLA-4 suggesting it is biologically active. All samples also demonstrated biologic activity for GPI-IL-12 as tested using a reporter cell line for IL-12 and displayed acceptable endotoxin levels in a range of 1-34 EU/mg TMV vaccine. The remaining 6 tumor samples are being processed. RNA-seq data from mouse model suggests that the TMV vaccine showed synergistic activity when combined with anti-PD1 antibody, with enhanced T cell responses. Conclusions: TMV vaccine production is feasible for a phase 1 clinical trial for patients whose resected tumor is at least 0.5 g in weight and 40% cellularity. Data in mice show that TMV vaccine synergizes with anti-PD1 in inducing tumor responses.

CanCellVar: A database for single-cell variants map in human cancer

Numerous variants, including both single-nucleotide variants (SNVs) in DNA and A>G RNA edits in mRNA as essential drivers of cellular proliferation and tumorigenesis, are commonly associated with cancer progression and growth. Thus, mining and summarizing single-cell variants will provide a refined and higher-resolution view of cancer and further contribute to precision medicine. Here, we established a database, CanCellVar, which aims to provide and visualize the comprehensive atlas of single-cell variants in tumor microenvironment. The current CanCellVar identified ∼3 million variants (∼1.4 million SNVs and ∼1.4 million A>G RNA edits) involved in 2,754,531 cells of 5 major cell types across 37 cancer types. CanCellVar provides the basic annotation information as well as cellular and molecular function properties of variants. In addition, the clinical relevance of variants can be obtained including tumor grade, treatment, metastasis, and others. Several flexible tools were also developed to aid retrieval and to analyze cell-cell interactions, gene expression, cell-development trajectories, regulation, and molecular structure affected by variants. Collectively, CanCellVar will serve as a valuable resource for investigating the functions and characteristics of single-cell variations and their roles in human tumor evolution and treatment.

An exhaustive pre-clinical study of a circRNA drug candidate (cmRNA1210) encoding IL-12sc for anti-tumor therapeutics.

e14569 Background: Circular RNA as emerging technology hasn't been approved of acceptance of any IND application. What’s the pharmacokinetics, pharmacodynamics and toxicity study of circular RNA drug candidate would be like is still unpublished. Here we provide an exhaustive pre-clinical study of a novel circular RNA drug candidate encoding IL-12sc, the immune stimulatory effector. Methods: Here, we performed an exhaustive pre-clinical study of a circular RNA drug candidate(cmRNA1210) encoding IL-12sc, including pharmacokinetics, pharmacodynamics, pharmacology and toxicology study. Meanwhile, we explored the bioactivity and cross reactivity of cmRNA1210 in vitro and the pharmacokinetics in vivo. Results: cmRNA1210 and its replace molecule exhibits robust therapeutic effect and good safety in syngeneic mouse tumor model and humanized human tumor mouse model. In situ administration of cmRNA1210 achieves abscopal effect and inhibits lung metastasis progression. Treatment with cmRNA1210 leads to cytokines change in plasma and tumor, cytotoxic immune cells accumulation in tumor and tumor tissue necrosis. Dynamic transcriptome profiling of tumor tissue post cmRNA1210 treatment. Combination with checkpoint inhibitor exhibits synergy effect and significantly decreases tumor relapse rate. Kaplan-Meier survival curve in TCGA dataset shows strong correlation of high IL-12A/B expression and long survival. Conclusions: The bioactivity and cross reactivity in rodents and primates of cmRNA1210 were validated in vitro. The advantage of pharmacokinetics of circular RNA was confirmed in vivo. cmRNA1210 exhibits robust anti-tumor effect in various tumor models, indicating its wild application situation in clinical. In situ treatment achieves abscopal effect and significantly inhibits lung metastasis progression. Systemic anti-tumor effect has been successfully initiated. The pharmacodynamics study reveals there are cytokine changes, immune cells activation or accumulation at tumor site and tumor tissue necrosis after cmRNA1210 treatment. cmRNA1210 as immune stimulatory effector works synergistically with checkpoint inhibitor. Kaplan-Meier survival analysis of IL-12 expression based on TCGA dataset shows there is a strong correlation of high expression and long survival. Overall, this pre-clinical study provides elaborate results of cmRNA1210 as anti-tumor therapeutics and demonstrates the great potential to enter clinical trial in the future.

TROCEPT: An oncolytic virus platform engineered for αvß6 integrin targeted tumor-localised expression of an immune checkpoint inhibitor following intravenous delivery.

e14579 Background: TROCEPT is a novel tumor-selective replicating, oncolytic adenovirus type 5 (Ad5) rationally engineered to remove all natural tissue tropisms. This engineering addresses the main limitation of other viral therapies which infect normal tissues and are rapidly removed by the liver and are therefore limited to local delivery. TROCEPT has been further engineered to specifically bind to αvβ6 integrin which is expressed at high frequency in the majority of epithelial cancers, so that following intravenous delivery the virus targets tumor tissue where it replicates and amplifies. In addition, the TROCEPT platform encodes transgenes which enables in-tumor production of powerful therapeutic drugs. The lead programme, TROCEPT-01, encodes a fully human, full length immune checkpoint inhibitor (ICI) antibody. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications. Methods: Proof of concept studies, both in vitro and in vivo, to demonstrate TROCEPT-01’s tumor selectivity and toxicity profile have been performed. Results: In vitro experiments demonstrate that viral replication, transgene expression and oncolytic cell death following infection with TROCEPT-01 is highly selective for αvβ6 integrin positive tumor cells compared to a panel of normal human primary cells. Intravenous delivery in in vivo SKOV3 ovarian human tumor xenograft mouse models demonstrate virus delivery, replication, and transgene expression in the tumor. In comparison to the parental Ad5 virus, the engineering of TROCEPT-01 leads to more viral delivery to the tumour, less delivery to normal tissue including liver, reduced markers of liver toxicity and reduced inflammatory cytokine release, all of which validate the tumour selectivity and safety profile of the virus. Conclusions: We have demonstrated that TROCEPT engineering enables highly selective tumor targeting and significant tumor exposure following intravenous delivery. TROCEPT-01’s in-tumor selective expression of ICIs enables high local drug concentration in the tumor, potentially reducing systemic toxicity and increasing efficacy. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications.

MOSAIC: An Artificial Intelligence-Based Framework for Multimodal Analysis, Classification, and Personalized Prognostic Assessment in Rare Cancers.

Rare cancers constitute over 20% of human neoplasms, often affecting patients with unmet medical needs. The development of effective classification and prognostication systems is crucial to improve the decision-making process and drive innovative treatment strategies. We have created and implemented MOSAIC, an artificial intelligence (AI)-based framework designed for multimodal analysis, classification, and personalized prognostic assessment in rare cancers. Clinical validation was performed on myelodysplastic syndrome (MDS), a rare hematologic cancer with clinical and genomic heterogeneities. We analyzed 4,427 patients with MDS divided into training and validation cohorts. Deep learning methods were applied to integrate and impute clinical/genomic features. Clustering was performed by combining Uniform Manifold Approximation and Projection for Dimension Reduction + Hierarchical Density-Based Spatial Clustering of Applications with Noise (UMAP + HDBSCAN) methods, compared with the conventional Hierarchical Dirichlet Process (HDP). Linear and AI-based nonlinear approaches were compared for survival prediction. Explainable AI (Shapley Additive Explanations approach [SHAP]) and federated learning were used to improve the interpretation and the performance of the clinical models, integrating them into distributed infrastructure. UMAP + HDBSCAN clustering obtained a more granular patient stratification, achieving a higher average silhouette coefficient (0.16) with respect to HDP (0.01) and higher balanced accuracy in cluster classification by Random Forest (92.7% ± 1.3% and 85.8% ± 0.8%). AI methods for survival prediction outperform conventional statistical techniques and the reference prognostic tool for MDS. Nonlinear Gradient Boosting Survival stands in the internal (Concordance-Index [C-Index], 0.77; SD, 0.01) and external validation (C-Index, 0.74; SD, 0.02). SHAP analysis revealed that similar features drove patients' subgroups and outcomes in both training and validation cohorts. Federated implementation improved the accuracy of developed models. MOSAIC provides an explainable and robust framework to optimize classification and prognostic assessment of rare cancers. AI-based approaches demonstrated superior accuracy in capturing genomic similarities and providing individual prognostic information compared with conventional statistical methods. Its federated implementation ensures broad clinical application, guaranteeing high performance and data protection.

Ionizing Radiation-treated NRF2-deficient Cancer Cells Promote Type I Interferon Signaling in Immune Cells.

Nuclear factor erythroid-derived 2-related factor-2 (NRF2) is a transcription factor that regulates stress response genes. It negatively regulates the immune system by acting as a transcriptional repressor of inflammatory genes or suppressing type I interferon (IFN) production pathways. NRF2 is often over-expressed in some tumors, including non-small cell lung cancer, and modulates these tumors via an immune-cold microenvironment. Thus, strategies to convert cold tumors into hot tumors are effective for cancer treatment. NRF2 was knocked-down or over-expressed in human cancer cells (A549, HeLa, H1299, H1650) and mouse mammary adenocarcinoma TS/A cells. Cells were irradiated or transfected with poly(I:C), and changes in type I IFN levels were examined using quantitative real-time polymerase chain reaction and western blotting. Cytosolic DNA was assayed via PicoGreen staining and immune and cancer cells were co-cultured. Regulation of NRF2 expression altered type I IFN levels in the human lung cancer cell line A549 and several solid tumors. Down-regulation of NRF2 resulted in increased levels of cytosolic DNA and activated the cGAS-STING pathway. We confirmed that type I IFN was induced in NRF2-down-regulated tumor cells using ionizing radiation (IR). Furthermore, when dendritic cells and macrophages were co-cultured with IR-exposed NRF2 knockdown tumor cells, the immune cells produced more IFNB1 and CXCL10. The immunosuppressive tumor cell environment is improved by NRF2 down-regulation, and IR treatment may promote immune cell signaling activation.

ONECUT2 is a druggable driver of luminal to basal breast cancer plasticity.

Tumor heterogeneity complicates patient treatment and can be due to transitioning of cancer cells across phenotypic cell states. This process is associated with the acquisition of independence from an oncogenic driver, such as the estrogen receptor (ER) in breast cancer (BC), resulting in tumor progression, therapeutic failure and metastatic spread. The transcription factor ONECUT2 (OC2) has been shown to be a master regulator protein of metastatic castration-resistant prostate cancer (mCRPC) tumors that promotes lineage plasticity to a drug-resistant neuroendocrine (NEPC) phenotype. Here, we investigate the role of OC2 in the dynamic conversion between different molecular subtypes in BC. We analyze OC2 expression and clinical significance in BC using public databases and immunohistochemical staining. In vitro, we perform RNA-Seq, RT-qPCR and western-blot after OC2 enforced expression. We also assess cellular effects of OC2 silencing and inhibition with a drug-like small molecule in vitro and in vivo. OC2 is highly expressed in a substantial subset of hormone receptor negative human BC tumors and tamoxifen-resistant models, and is associated with poor clinical outcome, lymph node metastasis and heightened clinical stage. OC2 inhibits ER expression and activity, suppresses a gene expression program associated with luminal differentiation and activates a basal-like state at the gene expression level. We also show that OC2 is required for cell growth and survival in metastatic BC models and that it can be targeted with a small molecule inhibitor providing a novel therapeutic strategy for patients with OC2 active tumors. The transcription factor OC2 is a driver of BC heterogeneity and a potential drug target in distinct cell states within the breast tumors.

Delivering Effector T Lymphocytes into Mice Carrying Human Brain Tumors

Delivering Effector T Lymphocytes into Mice Carrying Human Brain Tumors

Induction of fibrosis following exposure to bisphenol A and its analogues in 3D human uterine leiomyoma cultures

Bisphenol A (BPA) and its analogues (BPAF, BPS) are ubiquitous environmental contaminants used as plastic additives in various daily life products, with many concerns on their role as environmental estrogens. Uterine leiomyomas (fibroids) are highly prevalent gynecologic tumors with progressive fibrosis. Fibroids are hormone-responsive and may be the target of environmental estrogens. However, the effects of BPA, BPAF, and BPS exposure on uterine fibrosis are largely unknown. Here, we evaluated fibrosis and the crucial role of TGF-beta signaling in human fibroid tumors, the profibrotic effects of BPA, BPAF or BPS in a human 3D uterine leiomyoma (ht-UtLM) in vitro model, and the long-term outcomes of BPAF exposure in rat uterus. In 3D ht-UtLM spheroids, BPA, BPAF, and BPS all promoted cell proliferation and fibrosis by increasing the production of extracellular matrices. Further mechanistic analysis showed the profibrotic effects were induced by TGF-beta signaling activation mainly through SMAD2/3 pathway and crosstalk with multiple non-SMAD pathways. Furthermore, the profibrotic effects of BPAF were supported by observation of uterine fibrosis in vivo in rats following long-term BPAF exposure. Overall, the 3D ht-UtLM spheroid can be an important model for investigating environment-induced fibrosis in uterine fibroids. BPA and its analogues can induce fibrosis via TGF-beta signaling.

FK228 suppress the growth of human malignant pleural mesothelioma tumor independent to epithelioid or non-epithelioid histology

Human malignant pleural mesothelioma (hMPM) is an aggressive, rare disease with a poor prognosis. Histologically, MPM is categorized into epithelioid, biphasic, and sarcomatoid subtypes, with the epithelioid subtype generally displaying a better response to treatment. Conversely, effective therapies for the non-epithelioid subtypes are limited. This study aimed to investigate the potential role of FK228, a histone deacetylase inhibitor, in the suppression of hMPM tumor growth. We conducted a comprehensive analysis of the histological and molecular characteristics of two MPM cell lines, CRL-5820 (epithelioid) and CRL-5946 (non-epithelioid). CRL-5946 cells and non-epithelioid patient-derived xenografted mice exhibited heightened growth rates compared to those with epithelioid MPM. Both CRL-5946 cells and non-epithelioid mice displayed a poor response to cisplatin. However, FK228 markedly inhibited the growth of both epithelioid and non-epithelioid tumor cells in vitro and in vivo. Cell cycle analysis revealed FK228-induced G1/S and mitotic arrest in MPM cells. Caspase inhibitor experiments demonstrated that FK228-triggered apoptosis occurred via a caspase-dependent pathway in CRL-5946 but not in CRL-5820 cells. Additionally, a cytokine array analysis showed that FK228 reduced the release of growth factors, including platelet-derived and vascular endothelial growth factors, specifically in CRL-5946 cells. These results indicate that FK228 exhibits therapeutic potential in MPM by inducing cytotoxicity and modulating the tumor microenvironment, potentially benefiting both epithelioid and non-epithelioid subtypes.

Anticancer potential of four triterpenoids against NCI-60 human tumor cell lines

BackgroundThe burden of cancer incidences and mortality is rapidly increasing worldwide resulting in an increased demand for new therapies. Secondary metabolites extracted from medicinal plants have significantly contributed toward discovery of new cancer therapies some of which are in clinical use. In this study, anticancer potential of four triterpenoids, namely oleanonic acid (EK-2), 3-epi-oleanolic acid (EK-8), 1,2,3,22,23-pentahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (EK-4) and 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (EK-9), extracted from Ekebergia capensis Sparrm root bark was evaluated.ResultsWe employed CLC-Pred to initially evaluate cytotoxicity of previously isolated compounds in silico where predictions revealed high probability of bioactivity. The compounds were then submitted to the National Cancer Institute (NCI), Developmental Therapeutics Program, for bioactivity evaluation against NCI-60 human tumor cell lines. The four compounds demonstrated a range of potencies at a concentration of 10 µM. The results revealed that EK-9 was the most potent with mean growth percent of 32.84 and cases of lethality (negative growth percent) against two leukemia cell lines (HL-60 (TB) and RPMI-8226) and HT29 (colon cancer) and SK-MEL-5 (melanoma). This molecule was further evaluated in a five-dose assay where notable growth inhibition against leukemia cells, HL-60 (TB), RPMI-8226 and K-562 was observed with growth inhibitory activity (GI50) values of 3.10, 3.74 and 5.07 µM, respectively. In addition, total growth inhibition was observed at 11.2 μM and 18.9 μM for HL-60 (TB) and RPMI-8226 cells, respectively, partly accounting for the negative growth percent.ConclusionThe study has demonstrated anticancer properties of the four triterpenoids with compound EK-9 being the most potent overall having selective bioactivity in leukemia and breast cancer cells. Further studies focusing on elucidating its mechanism of action will be useful in exploration of the therapeutic potential of triterpenoids in general.

Comprehensive analysis to identify PUS7 as a prognostic biomarker from pan-cancer analysis to osteosarcoma validation.

Pseudouridylation has demonstrated the potential to control the development of numerous malignancies. PUS7(Pseudouridine Synthase 7) is one of the pseudouridine synthases, but the literature on this enzyme is limited to several cancer types. Currently, no investigation has been performed on the systematic pan-cancer analysis concerning PUS7 role in cancer diagnosis and prognosis. Employing public databases, including The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression Project (GTEx), Human Protein Atlas (HPA), UALCAN and Tumor Immune Single-cell Hub (TISCH), this work investigated the PUS7 carcinogenesis in pan-cancer. Differential expression analysis, prognostic survival analysis and biological function were systematically performed. Furthermore, PUS7 potential as an osteosarcoma biomarker for diagnosis and prognosis was assessed in this study. The findings indicated that PUS7 was overexpressed in the majority of malignancies. High PUS7 expression contributed to the poor prognosis among 11 cancer types, including Adrenocortical Cancer (ACC), Bladder Cancer (BLCA), Liver Cancer (LIHC), Kidney Papillary Cell Carcinoma (KIRP), Mesothelioma (MESO), Lower Grade Glioma (LGG), Kidney Chromophobe (KICH), Sarcoma (SARC), osteosarcoma (OS), Pancreatic Cancer (PAAD), and Thyroid Cancer (THCA). In addition, elevated PUS7 expression was linked to advanced TNM across multiple malignancies, including ACC, BLCA, KIRP, LIHC and PAAD. The function enrichment analysis revealed that PUS7 participates in E2F targets, G2M checkpoint, ribosome biogenesis, and rRNA metabolic process. Moreover, PUS7 is also a reliable biomarker and a potential therapeutic target for osteosarcoma. In summary, PUS7 is a putative pan-cancer biomarker that reliably forecasts cancer patients' prognosis. In addition, this enzyme regulates the cell cycle, ribosome biogenesis, and rRNA metabolism. Most importantly, PUS7 possibly regulates osteosarcoma initiation and progression.