Patient-derived Xenograft Mouse Research Articles

P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

Glioblastoma stem cells (GSCs) are pivotal in the recurrence and drug resistance of glioblastoma multiforme (GBM). However, precision therapeutic and diagnostic markers for GSCs have not been fully established. Here, using bioinformatics and experimental analysis, we identified P4HB, a protein disulfide isomerase, as a serum marker that maintains stemness in GSCs through the Wnt/β-catenin signaling pathway. Transcriptional silencing of P4HB induces apoptosis and diminishes stem cell-like characteristics in GSCs. Treatments with the chemical CCF624 or the China National Medical Products Administration (NMPA)-approved securinine significantly prolonged survival in patient-derived xenograft mouse models, underscoring P4HB’s potential as a therapeutic target and presenting an expedited path to clinical application through drug repurposing. Additionally, elevated P4HB levels in patient serum were found to correlate with disease progression, underscoring its utility as a biomarker and its promise for precision medicine.

Oncolytic virus encoding 4-1BBL and IL15 enhances the efficacy of tumor-infiltrating lymphocyte adoptive therapy in HCC.

Previous studies have found that oncolytic virus (OVs) can improve the efficacy of TIL adoptive therapy in oral cancer, colon cancer, and pancreatic cancer. However, the curative effect in hepatocellular carcinoma (HCC) is still unclear. Therefore, this study aims to explore the therapeutic effect and mechanism of OVs encoding 4-1BBL and IL15 (OV-4-1BBL/IL15) combined with TIL adoptive therapy on HCC. In this study, the role and immunological mechanism of armed OVs combined with TILs were evaluated by flow cytometry and ELISA in patient-derived xenograft and syngeneic mouse tumor models. Co-culturing with TILs can up-regulate the expression of antigen-presenting cell (APC) markers on the surface of OV-infected primary HCC cells, and promote the specific activation ability and tumor-killing ability of TILs. OV-4-1BBL/IL15 combined with TIL adoptive therapy could induce tumor volume reduction and anti-tumor immune memory in patient-derived xenograft and syngeneic mouse tumor models. Furthermore, OV combined with TIL adoptive therapy can endow tumor cells with aAPC characteristics, activate T cells at the same time, and reprogram tumor macrophages into anti-tumor phenotype. OV-4-1BBL/IL15 can stimulate the anti-tumor potential of TIL therapy in HCC, and possess broad clinical application prospects.

Generation of chimeric antigen receptor T cells targeting p95HER2 in solid tumors

The redirection of T lymphocytes against tumor-associated or tumor-specific antigens, using bispecific antibodies or chimeric antigen receptors (CAR), has shown therapeutic success against certain hematological malignancies. However, this strategy has not been effective against solid tumors. Here, we describe the development of CAR T cells targeting p95HER2, a tumor-specific antigen found in HER2-amplified solid tumors. These CAR T cells display robust activity against p95HER2-expressing cell lines but demonstrate limited efficacy against patient-derived xenografts. As p95HER2 is invariably detectable on tumor cells that overexpress HER2, but not those that express HER2 at normal levels, we arm p95HER2-specific CAR T cells with affinity-tuned bispecific antibodies against HER2 and CD3 in order to redirect them only to HER2-amplified cells. The combination of p95HER2.CAR T cells and HER2 x CD3 bispecific antibodies lead to a complete regression in three HER2-positive, patient-derived mouse xenografts tumor models. This combination represents a promising strategy to redirect T cells against a subset of HER2-positive tumors.

Integrated proteomics and scRNA-seq analyses of ovarian cancer reveal molecular subtype-associated cell landscapes and immunotherapy targets.

Epithelial ovarian cancer (EOC) represents the most lethal gynaecological malignancy, yet understanding the connections between its molecular subtypes and their therapeutic implications remains incomplete. We conducted mass spectrometry-based proteomics analyses of 154 EOC tumour samples and 29 normal fallopian tubes, and single-cell RNA sequencing (scRNA-seq) analyses of an additional eight EOC tumours to classify proteomic subtypes and assess their cellular ecosystems and clinical significance. The efficacy of identified therapeutic targets was evaluated in patient-derived xenograft (PDX) and orthotopic mouse models. We identified four proteomic subtypes with distinct clinical relevance: malignant proliferative (C1), immune infiltrating (C2), Fallopian-like (C3) and differentiated (C4) subtypes. C2 subtype was characterized by lymphocyte infiltration, notably an increased presence of GZMK CD8+ T cells and phagocytosis-like MRC+ macrophages. Additionally, we identified CD40 as a specific prognostic factor for C2 subtype. The interaction between CD40+ phagocytosis-like macrophages and CD40RL+ IL17R CD4+ T cells was correlated with a favourable prognosis. Finally, we established a druggable landscape for non-immune EOC patients and verified a TYMP inhibitor as a promising therapeutic strategy. Our study refines the current immune subtype for EOC, highlighting CD40 agonists as promising therapies for C2 subtype patients and targeting TYMP for non-immune patients.

CD40 agonist on patient-derived xenograft mice for the treatment of B-cell acute lymphoblastic leukemia.

Cluster of differentiation 40 (CD40) is expressed on B-cell acute lymphoblastic leukemia (B-ALL) cases. However, the effect of CD40 activation on B-ALL cells has never been tested in vivo. The aim of our preclinical study was to investigate the therapeutic potential of a CD40 agonist in the treatment of B-ALL using patient-derived xenograft (PDX) mouse models. Intravenous administration of the CD40 agonist significantly impeded B-ALL cell proliferation and growth in vivo, accompanied by rapid activation of the extracellular signal-regulated kinase (ERK) pathway, leading to the induction of apoptosis and disruption of cell cycle progression. Co-treatment with a specific inhibitor of ERK further demonstrated that CD40 stimulation induced the pro-apoptosis of B-ALL cells in an ERK-dependent manner. Proteomic analysis revealed alterations in key signaling pathways associated with B-ALL expansion and maintenance. Moreover, the CD40 agonist markedly reduced the frequency of leukemia-initiating cells and leukemia development in PDX mice. Our study showed that the CD40 agonist can be associated with chemotherapeutic agents such as vincristine and dexamethasone, and this combination showed improved effectiveness. Additionally, the CD40 agonist was more effective on pre-B-ALL (EGIL B-III) that expressed CD40, than on common B-ALL (EGIL B-II) that lacked CD40 expression. These findings suggest that CD40 agonists are promising immunotherapeutic candidates for pediatric B-ALL, warranting further clinical investigations to improve patient outcomes in CD40-expressing B-ALL.

TMOD-08. DETAILED GENERATION OF AN ORTHOTOPIC INTRACRANIAL GLIOBLASTOMA (GBM) PATIENT-DERIVED XENOGRAFT (PDX) MODEL FOR PRECLINICAL IMMUNOTHERAPEUTIC STUDIES

Abstract PURPOSE This study aimed to establish an orthotopic intracranial PDX mouse model using cryopreserved GBM tissues expanded in a heterotopic subcutaneous PDX model for immunotherapeutic studies. The model is designed to closely replicate human GBM, enabling the exploration of its pathophysiology and the development of effective immunotherapies for this type of cancer. METHODS Glioblastoma tissues from 16 fresh and cryopreserved samples were used to establish a heterotopic subcutaneous PDX mouse model for tumor expansion. Successful engraftments were then used to create an orthotopic intracranial PDX mouse model. The tumor formation rates and durations were estimated in both models. Tumor fidelity to the original GBM tumors was confirmed through immunohistology (GFAP, P53, and Ki67) and genetic analysis (whole exome sequencing, RNA sequencing, and methylome analysis). The therapeutic effects of immunotherapy (allogenic NK cells) in combination with Avastin and Irinotecan were tested in the orthotopic intracranial PDX model. RESULTS The orthotopic intracranial GBM mouse model achieved 100% engraftment from successful tumor growth in the subcutaneous PDX model. The overall success rate was 81.25% (85.71% for cryopreserved tissues), with a median process duration of 26.5 weeks (19 weeks for the subcutaneous and 7.5 weeks for intracranial establishment). No significant differences in tumor formation were observed within one year for cryopreserved tissue in the subcutaneous model. Molecular and genetic analysis confirmed similar tumor characteristics between the intracranial PDX tumors and the original patient tumors. Additionally, NK cells combined with Avastin and Irinotecan demonstrated efficacy in this model. CONCLUSIONS We successfully established an orthotopic intracranial PDX mouse model using cryopreserved GBM tissues, with no observed differences within one year. These models accurately mirror patients’ tumor characteristics, promising advancements in glioblastoma research and treatment, particularly for immunotherapy studies. Keywords: Glioblastoma (GBM), cryopreserved GBM tissues, patient-derived xenograft (PDX) model, NOD/SCID IL-12Rγnull (NSG) mice

SLC7A9 suppression increases chemosensitivity by inducing ferroptosis via the inhibition of cystine transport in gastric cancer

SLC7A9 is responsible for the exchange of dibasic amino acids and cystine (influx) for neutral amino acids (efflux). Cystine/cysteine transport is related to ferroptosis. Sanger sequencing detected TP53 status of cancer cells. Transcriptomic sequencing and untargeted metabolome profiling were used to identify differentially expressed genes and metabolites, respectively, upon SLC7A9 overexpression. CCK8, cell clonality, and EdU assays were used to observe cell proliferation. Cystine probes, glutathione (GSH) probes, and lipid ROS probes were used to examine cystine, GSH, and lipid ROS levels. 13C metabolic flow assays were used to monitor cellular cystine and GSH metabolism. Patient-derived organoids (PDO), immunocompetent MFC mice allograft models and patient-derived xenograft (PDX) models were used to evaluate SLC7A9 impact on chemotherapeutic response and to observe therapeutic effect of SLC7A9 knockdown. Elevated SLC7A9 expression levels in gastric cancer cells were attributed to p53 loss. SLC7A9 knockdown suppressed the proliferation and increased the chemotherapy sensitivity of the cells. Chemotherapy was more effective in PDX and immunocompetent mice models upon SLC7A9 knockdown. Differentially expressed genes and metabolites between the SLC7A9 overexpression and control groups were associated with ferroptosis and GSH metabolism. SLC7A9 knockdown reduced cystine transport into cells, hampered intracellular cystine and GSH metabolic flow, decreased GSH synthesis, and increased lipid ROS levels in gastric cancer cells. Erastin was more effective at inducing ferroptosis in PDO and PDX models upon SLC7A9 knockdown. SLC7A9 promotes gastric cancer progression by acting as a suppressor of ferroptosis, independent of SLC7A11, which is negatively regulated by p53. This work was supported by National Natural Science Foundation of China, Innovation Promotion Program of NHC and Shanghai Key Labs SIBPT, and Shanghai Academy of Science & Technology.

Telaglenastat as an alternative to cisplatin as a radiosensitizer in the treatment of head and neck squamous cell carcinoma

The efficacy of radiation treatment (RT) of head and neck squamous cell carcinoma (HNSCC) is limited by radioresistance and the toxicity of FDA approved radiosensitizers. In extension to our previous research where we demonstrated that telaglenastat (CB839) increased efficacy of RT in in vitro and in vivo HNSCC models, here, we examine the radiosensitizing effects of telaglenastat in comparison to cisplatin's, as cisplatin is currently the standard of care for concurrent therapy. Combination of telaglenastat with RT reduced tumor volume in a HNSCC patient derived xenograft mouse model. The efficacy of telaglenastat with RT in reducing cell survival and increasing apoptosis was similar if not greater than that of cisplatin with RT in Cal27 and HN5 HNSCC cells. The addition of telaglenastat increased reactive oxygen species and reduced the antioxidant glutathione in both Cal27 and HN5 cells. Reverse Phase Protein Array analyses revealed alterations in cell death and DNA damage response proteins. This study provides the scientific underpinnings for the use of telaglenastat as a radiosensitizer in the treatment of HNSCC either as an alternative to cisplatin or in cisplatin-ineligible patients.

Overcoming multi-drug resistance in SCLC: a synergistic approach with venetoclax and hydroxychloroquine targeting the lncRNA LYPLAL1-DT/BCL2/BECN1 pathway

BackgroundSmall cell lung cancer (SCLC) stands as one of the most lethal malignancies, characterized by a grim diagnosis and prognosis. The emergence of multi-drug resistance poses a significant hurdle to effective therapy. Although previous studies have implicated the long noncoding RNA LYPLAL1-DT in the tumorigenesis of SCLC, the precise role of the highly expressed LYPLAL1-DT in SCLC chemoresistance and the underlying mechanism remain inadequately understood.MethodscDDP-, VP-16- and PTX-resistant SCLC cells lines were established. The viabilities of SCLC cells were assessed by CCK-8 assay in vitro and xenograft tumor formation assay in vivo. Apoptosis was evaluated by FACS, Western blot and JC-1 fluorescence staining, while autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of LYPLAL1-DT were further investigated by gain- and loss-of-function assays in vitro. Furthermore, the therapeutic efficacy of the combination of venetoclax and HCQ with cDDP, VP-16 or PTX was evaluated by cell line, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice model.ResultsOur findings revealed that LYPLAL1-DT is upregulated in chemoresistant SCLC cell lines. Gain- and loss-of-function assays demonstrated that LYPLAL1-DT impairs sensitivity to cDDP, VP-16, or PTX both in vitro and in vivo. Overexpression of LYPLAL1-DT significantly enhanced autophagy and inhibited apoptosis in SCLC cells. Further analyses, including RIP and RNA pull-down assays, revealed that LYPLAL1-DT promotes the expression of BCL2 by sponging miR-204-5p and is implicated in the assembly of the autophagy-specific complex (BECN1/PtdIns3K complex). Combining venetoclax and HCQ with cDDP, VP-16, or PTX effectively mitigated chemoresistance in SCLC cells and suppressed tumor growth in CDX and PDX models without inducing obvious toxic effects.ConclusionsOur findings demonstrate that upregulation of LYPLAL1-DT sequesters apoptosis through the LYPLAL1-DT/miR-204-5p/BCL2 axis and promotes autophagy by facilitating the assembly of the BECN1/PtdIns3K complex, thereby mediating multi-drug resistance of SCLC. The triple combination of venetoclax, HCQ, in conjunction with cDDP, VP-16 or PTX overcomes refractory SCLC, shedding light on a potential therapeutic target for combating SCLC chemoresistance.

Tumor-Associated Tractography Derived from High-Angular-Resolution Q-Space MRI May Predict Patterns of Cellular Invasion in Glioblastoma.

The invasion of glioblastoma cells beyond the visible tumor margin depicted by conventional neuroimaging is believed to mediate recurrence and predict poor survival. Radiomic biomarkers that are associated with the direction and extent of tumor infiltration are, however, non-existent. Patients from a single center with newly diagnosed glioblastoma (n = 7) underwent preoperative Q-space magnetic resonance imaging (QSI; 3T, 64 gradient directions, b = 1000 s/mm2) between 2018 and 2019. Tumors were manually segmented, and patterns of inter-voxel coherence spatially intersecting each segmentation were generated to represent tumor-associated tractography. One patient additionally underwent regional biopsy of diffusion tract- versus non-tract-associated tissue during tumor resection for RNA sequencing. Imaging data from this cohort were compared with a historical cohort of n = 66 glioblastoma patients who underwent similar QSI scans. Associations of tractography-derived metrics with survival were assessed using t-tests, linear regression, and Kaplan-Meier statistics. Patient-derived glioblastoma xenograft (PDX) mice generated with the sub-hippocampal injection of human-derived glioblastoma stem cells (GSCs) were scanned under high-field conditions (QSI, 7T, 512 gradient directions), and tumor-associated tractography was compared with the 3D microscopic reconstruction of immunostained GSCs. In the principal enrollment cohort of patients with glioblastoma, all cases displayed tractography patterns with tumor-intersecting tract bundles extending into brain parenchyma, a phenotype which was reproduced in PDX mice as well as in a larger comparison cohort of glioblastoma patients (n = 66), when applying similar methods. Reconstructed spatial patterns of GSCs in PDX mice closely mirrored tumor-associated tractography. On a Kaplan-Meier survival analysis of n = 66 patients, the calculated intra-tumoral mean diffusivity predicted the overall survival (p = 0.037), as did tractography-associated features including mean tract length (p = 0.039) and mean projecting tract length (p = 0.022). The RNA sequencing of human tissue samples (n = 13 tumor samples from a single patient) revealed the overexpression of transcripts which regulate cell motility in tract-associated samples. QSI discriminates tumor-specific patterns of inter-voxel coherence believed to represent white matter pathways which may be susceptible to glioblastoma invasion. These findings may lay the groundwork for future work on therapeutic targeting, patient stratification, and prognosis in glioblastoma.

Preclinical Multi-Omic Assessment of Pioglitazone in Skeletal Muscles of Mice Implanted with Human HER2/neu Overexpressing Breast Cancer Xenografts.

Background/Objectives: Breast cancer (BC) is the second most commonly diagnosed cancer worldwide and is accompanied by fatigue during both active disease and remission in the majority of cases. Our lab has measured fatigue in isolated muscles from treatment-naive BC patient-derived orthotopic xenograft (BC-PDOX) mice. Here, we conducted a preclinical trial of pioglitazone in BC-PDOX mice to determine its efficacy in ameliorating BC-induced muscle fatigue, as well as its effects on transcriptomic, metabolomic, and lipidomic profiles in skeletal muscle. Methods: The pioglitazone and vehicle groups were treated orally for 4 weeks upon reaching a tumor volume of 600 mm3. Whole-animal indirect calorimetry was used to evaluate systemic metabolic states. The transcriptome was profiled using short-read bulk RNA sequencing (RNA-seq). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to profile the metabolome and lipidome. Fast and slow skeletal muscle function were evaluated using isolated ex vivo testing. Results: Pioglitazone was associated with a 16.634% lower average O2 consumption (mL∙h-1, p = 0.035), 16.309% lower average CO2 production (mL∙h-1, p = 0.022), and 16.4% lower cumulative energy expenditure (EE) (kcal∙h-1, p = 0.035), with no changes in substrate utilization. RNA-seq supported the downstream effects of pioglitazone on target genes and displayed considerable upregulation of mitochondrial bioenergetic pathways. K-means cluster 5 showed enrichment of the PPAR signaling pathway (adj. p < 0.05, Log2FC = 2.58). Skeletal muscle metabolomic and lipidomic profiles exhibited dysregulation in response to BC, which was partially restored in pioglitazone-treated mice compared to vehicle-treated BC-PDOX mice. In particular, the overall abundance of total ceramide levels was significantly lower in the PioTx group (-46.327%, p = 0.048). Despite molecular support for pioglitazone's efficacy, isolated muscle function was not affected by pioglitazone treatment. No significant difference in the area under the fatigue curve (AUC) was found between the pioglitazone and vehicle groups (p = 0.596). Conclusions: BC induces multi-omic dysregulation in skeletal muscle, which pioglitazone partially ameliorates. Future research should focus on profiling systemic metabolic dysfunction, identifying molecular biomarkers of fatigue, and testing alternative pioglitazone treatment regimens.

LAP2α orchestrates alternative lengthening of telomeres suppression through telomeric heterochromatin regulation with HDAC1: unveiling a potential therapeutic target

In response to the challenge of telomere attrition during DNA replication, cancer cells predominantly employ telomerase or, in 10–15% of cases, the alternative lengthening of telomeres (ALT). The intricate details of ALT, however, remain elusive. In this study, we unveil that the knockdown of lamina-associated polypeptide 2 alpha (LAP2α) in ALT cells results in telomere dysfunction, triggering a notable increase in ALT-associated hallmarks, including high frequencies of PML bodies (APBs), C-rich extrachromosomal circles (C-circles), and telomere sister chromatid exchange (T-SCE). Furthermore, LAP2α emerges as a crucial player in break-induced telomere replication for telomerase-positive cells following telomeric double-strand breaks. Mechanistically, our investigation suggests that LAP2α may influence the regulation of the heterochromatic state of telomeres, thereby affecting telomeric accessibility. In line with our findings, LAP2α expression is markedly reduced in ALT-positive osteosarcoma. And the use of methotrexate (MTX) can restore the heterochromatin state altered by LAP2α depletion. This is evidenced by a significant inhibition of tumor proliferation in ALT-positive patient-derived xenograft (PDX) mouse models. These results indicate the important role of LAP2α in regulating ALT activity and offer insights into the interplay between lamina-associated proteins and telomeres in maintaining telomere length. Importantly, our findings may help identify a more appropriate target population for the osteosarcoma therapeutic drug, MTX.

Development and Validation of a Comprehensive Prognostic and Depression Risk Index for Gastric Adenocarcinoma

Depressive disorder contributes to the initiation and prognosis of patients with cancer, but the interaction between cancer and depressive disorder remains unclear. We generated a gastric adenocarcinoma patient-derived xenograft mice model, treated with chronic unpredictable mild stimulation. Based on the RNA-sequence from the mouse model, patient data from TCGA, and MDD-related (major depressive disorder) genes from the GEO database, 56 hub genes were identified by the intersection of differential expression genes from the three datasets. Molecular subtypes and a prognostic signature were generated based on the 56 genes. A depressive mouse model was constructed to test the key changes in the signatures. The signature was constructed based on the NDUFA4L2, ANKRD45, and AQP3 genes. Patients with high risk-score had a worse overall survival than the patients with low scores, consistent with the results from the two GEO cohorts. The comprehensive results showed that a higher risk-score was correlated with higher levels of tumor immune exclusion, higher infiltration of M0 macrophages, M2 macrophages, and neutrophils, higher angiogenetic activities, and more enriched epithelial–mesenchymal transition signaling pathways. A higher risk score was correlated to a higher MDD score, elevated MDD-related cytokines, and the dysfunction of neurogenesis-related genes, and parts of these changes showed similar trends in the animal model. With the Genomics of Drug Sensitivity in Cancer database, we found that the gastric adenocarcinoma patients with high risk-score may be sensitive to Pazopanib, XMD8.85, Midostaurin, HG.6.64.1, Elesclomol, Linifanib, AP.24534, Roscovitine, Cytarabine, and Axitinib. The gene signature consisting of the NDUFA4L2, ANKRD45, and AQP3 genes is a promising biomarker to distinguish the prognosis, the molecular and immune characteristics, the depressive risk, and the therapy candidates for gastric adenocarcinoma patients.

The Role of CENPK Splice Variant in Abiraterone Response in Metastatic Castration-Resistant Prostate Cancer.

Most patients with metastatic prostate cancer eventually develop resistance to primary androgen deprivation therapy. To identify predictive biomarker for Abiraterone acetate/prednisone resistance, we screened alternative splice variants between responders and non-responders from the PROMOTE clinical study and pinned down the most significant variant, CENPK-delta8. Through preclinical patient-derived mouse xenograft (PDX) and 3D organoids obtained from responders and non-responders, as well as in vitro models, aberrant CENPK-delta8 expression was determined to link to drug resistance via enhanced migration and proliferation. The FLNA and FLOT1 were observed to specifically bind to CENK-delta8 rather than wild-type CENPK, underscoring the role of CENPK-delta8 in cytoskeleton organization and cell migration. Our study, leveraging data from the PROMOTE study, TCGA, and TCGA SpliceReq databases, highlights the important function of alternative splice variants in drug response and their potential to be prognostic biomarkers for improving individual therapeutic outcomes in precision medicine.

Three-dimensional analysis of mitochondria in a patient-derived xenograft model of triple negative breast cancer reveals mitochondrial network remodeling following chemotherapy treatments.

Mitochondria are hubs of metabolism and signaling and play an important role in tumorigenesis, therapeutic resistance, and metastasis in many cancer types. Various laboratory models of cancer demonstrate the extraordinary dynamics of mitochondrial structure, but little is known about the role of mitochondrial structure in resistance to anticancer therapy. We previously demonstrated the importance of mitochondrial structure and oxidative phosphorylation in the survival of chemotherapy-refractory triple negative breast cancer (TNBC) cells. As TNBC is a highly aggressive breast cancer subtype with few targeted therapy options, conventional chemotherapies remain the backbone of early TNBC treatment. Unfortunately, approximately 45% of TNBC patients retain substantial residual tumor burden following chemotherapy, associated with abysmal prognoses. Using an orthotopic patient-derived xenograft mouse model of human TNBC, we compared mitochondrial structures between treatment-naïve tumors and residual tumors after conventional chemotherapeutics were administered singly or in combination. We reconstructed 1,750 mitochondria in three dimensions from serial block-face scanning electron micrographs, providing unprecedented insights into the complexity and intra-tumoral heterogeneity of mitochondria in TNBC. Following exposure to carboplatin or docetaxel given individually, residual tumor mitochondria exhibited significant increases in mitochondrial complexity index, area, volume, perimeter, width, and length relative to treatment-naïve tumor mitochondria. In contrast, residual tumors exposed to those chemotherapies given in combination exhibited diminished mitochondrial structure changes. Further, we document extensive intra-tumoral heterogeneity of mitochondrial structure, especially prior to chemotherapeutic exposure. These results highlight the potential for structure-based monitoring of chemotherapeutic responses and reveal potential molecular mechanisms that underlie chemotherapeutic resistance in TNBC.

Enhancing efficacy of the MEK inhibitor trametinib with paclitaxel in KRAS -mutated colorectal cancer.

KRAS is frequently mutated in the tumors of patients with metastatic colorectal cancer (mCRC) and thus represents a valid target for therapy. However, the strategies of targeting KRAS directly and targeting the downstream effector mitogen-activated protein kinase kinase (MEK) via monotherapies have shown limited efficacy. Thus, there is a strong need for novel, effective combination therapies to improve MEK-inhibitor efficacy in patients with KRAS -mutated mCRC. Our objective was to identify novel drug combinations that enhance MEK-inhibitor efficacy in patients with KRAS -mutated mCRC. In this study, we performed unbiased high-throughput screening (HTS) to identify drugs that enhance the efficacy of MEK inhibitors in vitro , and we validated the efficacy of the drugs in vivo . HTS was performed using 3-dimensional CRC spheroids. Trametinib, the anchor drug, was probed with 2 clinically ready libraries of 252 drugs to identify effective drug combinations. The effects of the drug combinations on CRC cell proliferation and apoptosis were further validated using cell growth assays, flow cytometry, and biochemical assays. Proteomic and immunostaining studies were performed to determine the effects of the drugs on molecular signaling and cell division. The effects of the drug combinations were examined in vivo using CRC patient-derived xenografts. HTS identified paclitaxel as being synergistic with trametinib. In vitro validation showed that, compared with monotherapies, this drug combination demonstrated strong inhibition of cell growth, reduced colony formation, and enhanced apoptosis in multiple KRAS -mutated CRC cell lines. Mechanistically, combining trametinib with paclitaxel led to alterations in signaling mediators that block cell cycle progression and increases in microtubule stability that resulted in significantly higher defects in the mitosis. Finally, the combination of trametinib with paclitaxel exhibited significant inhibition of tumor growth in several KRAS -mutant patient-derived xenograft mouse models. Our data provide evidence supporting clinical trials of trametinib with paclitaxel as a novel therapeutic option for patients with KRAS -mutated, metastatic CRC.

Quercetin inhibits truncated isoform of dopamine- and cAMP-regulated phosphoprotein as adjuvant treatment for trastuzumab therapy resistance in HER2-positive breast cancer

Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer (BC). The truncated isoform of dopamine- and cAMP-regulated phosphoprotein (t-DARPP) has been reported to be involved in trastuzumab therapy resistance and promoting tumor progression. To evaluate the t-DARPP expression in BC, paired tumors and surrounding normal tissues were analyzed by real-time polymerase chain reaction and confirmed higher DARPP-32 kDa family mRNA expression in HER2+ BC tumor tissues. We established 2 patient-derived xenografts (PDX) mice models to test the efficacy of trastuzumab, named model 1 (non-responder) and model 2 (responder). t-DARPP and p95-HER2 protein-protein interactions were detected in PDX tumor tissue from non-responders using Förster resonance energy transfer assays. Instead, there is no response from the responder. Furthermore, mechanistic studies using transwell and Western blot assays demonstrated that t-DARPP could upregulate epithelial-mesenchymal transition signaling proteins, enhance p95-HER2 expression and promote cell migration. We found that quercetin effectively reduced t-DARPP expression in HER2+ BC cells. In t-DARPP ShRNA-suppressed cells, quercetin synergistically enhanced trastuzumab-induced apoptotic cell death and G2/M phase arrest. In conclusion, the combination of quercetin and trastuzumab treatment by targeting t-DARPP in HER2+ BC patients has the potential as a biomarker for mitigating drug resistance.

Brain-Targeted Cas12a Ribonucleoprotein Nanocapsules Enable Synergetic Gene Co-Editing Leading to Potent Inhibition of Orthotopic Glioblastoma.

Gene-editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene-editing-based therapy is unlikely to be successful; therefore, a multi-gene knockout strategy is preferred for effective GBM inhibition. Here, a non-invasive, biodegradable brain-targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half-life, efficient blood-brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient-derived GSC xenograft mouse models with negligible off-target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi-oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM.

Reserpine, a novel N6-methyladenosine regulator, reverses Lenvatinib resistance in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is an aggressive malignancy and a growing global health problem. Reserpine (Res), a plant-derived hypertension drug, has been reported to possess anti-tumor efficacy. However, the role and function of Res in N6-methyladenosine (m6A) regulation and Lenvatinib (Len) resistance in HCC have not been clarified. PurposeTo verify whether Res can be used as a natural small-molecule regulator of m6A to reverse Len resistance in HCC. MethodsDot blotting, Western blotting and m6A quantification were used to compare and analyze the differential expression of m6A and its methyltransferase METTL3. Western blotting, Real-Time PCR (RT-PCR), cellular thermal shift assay (CETSA) and molecular docking were used to explore the mechanism of interaction between Res and m6A. The effects of Res on the biological characteristics of Lenvatinib-resistant HCC cells were investigated through CCK-8, clone formation, and Transwell assays. Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models were used to assess the ability of Res to reverse Len resistance in vivo. MeRIP m6A sequencing, PATHWAY analysis and Western blotting were used to analyze the downstream signaling pathways and genes involved in Res-mediated reversal of Len resistance. ResultsLen resistance in HCC is related to the increased m6A level and the high expression of METTL3. Res affects the activity of METTL3 protein by binding to it, thereby downregulating the level of m6A. In vitro study showed that Res can sensitize HCC cells to the anti-tumor effects of Len treatment, including blocking proliferation, inhibiting migration, and inducing apoptosis. Len-resistant CDX and PDX models revealed that Res can reverse the resistant phenotype, with the tumor inhibition rates of 77.46 % and 62.1 %, respectively, when combined with Len treatment. Analysis of xenograft tissues showed that the combination of Res and Len down-regulates the m6A level, reduces proliferation biomarkers, and induces apoptosis, which is consistent with the in vitro data. Mechanistically, our preliminary results indicate that Res can up-regulate the SMAD3 level by down-regulating m6A in Len-resistant cells. ConclusionsReserpine, a small-molecule regulator of m6A, reverses Lenvatinib-resistant phenotypes, including proliferation, migration and anti-apoptosis, in vitro and in vivo by targeting SMAD3 and down-regulating the m6A level in HCC.

Prediction of the response to antiangiogenic sunitinib therapy by non-invasive hybrid diffuse optics in renal cell carcinoma.

In this work, broadband diffuse reflectance spectroscopy (DRS) and diffuse correlation spectroscopy (DCS) were used to quantify deep tissue hemodynamics in a patient-derived orthotopic xenograft mouse model of clear cell renal cancer undergoing antiangiogenic treatment. A cohort of twenty-two mice were treated with sunitinib and compared to thirteen control untreated mice, and monitored by DRS/DCS. A reduction in total hemoglobin concentration (THC, p = 0.03), oxygen saturation (SO2, p = 0.03) and blood flow index (BFI, p = 0.02) was observed over the treatment course. Early changes in tumor microvascular blood flow and total hemoglobin concentration were correlated with the final microvessel density (p = 0.014) and tumor weight (p = 0.024), respectively. Higher pre-treatment tumor microvascular blood flow was observed in non-responder mice with respect to responder mice, which was statistically predictive of the tumor intrinsic resistance (p = 0.01). This hybrid diffuse optical technique provides a method for predicting tumor intrinsic resistance to antiangiogenic therapy and could be used as predictive biomarker of response to antiangiogenic therapies in pre-clinical models.