Добірка наукової літератури з теми "Comparison with in vitro cultures and in vivo xenografts"

2072 Background: Human glioma stem cells and xenograft lines are common translational models in neuro-oncology but it has not been established if they are genetically and phenotypically comparable. This study aimed to determine if human glioma xenografts and stem cell-derived tumors had similar genotypes. Methods: Matched glioma stem cell cultures and subcutaneous xenograft lines were generated from four human glioblastoma specimens (BT114, BT116, BT120, BT132). Comparison was made between subcutaneous stem cell-derived tumors (flank) and xenografts established in nude mice. Copy number variation (CNV) and gene expression microarray studies were performed. Results: Various differences in copy number and gene expression were seen. Observed CNVs included regions within EGFR, myc, and p16 (INK). For example, EGFR copy number was two fold higher in xenografts vs. stem cell-derived tumor in one line (BT114). This difference was corroborated by western blot. Other differences included a heat shock protein homolog (DNAJA4), tetraspanin 13, and a p53 family target gene (ISG20L1). Two lines (BT114, BT116) had a greater than two fold increase in DNAJA4 expression in xenografts vs. stem cell-derived tumors (p = 0.04, 0.01). Two cell lines (BT116, BT120) had a two to eight fold increase in tetraspanin 13 expression in xenografts (p = 0.02, 0.05). However, neither copy number nor gene expression variations were consistent across all cell lines. Conclusions: Xenografts and glioma stem cell-derived tumors established from the same patient specimens have distinct genotypes. Further work is needed to establish if these differences are random or represent characteristic changes selected by different in vitro or in vivo pressures. However, these variations raise questions regarding which model is ideal for studying glioma biology, and which ones best replicate glioma characteristics in human patients.

351 Background: Despite recently approved novel agents, taxane-based chemotherapy remains the major therapeutic strategy for metastatic castration-refractory prostate cancer/mCRPC. Still mCRPC continues to be incurable. Besides, patients often experience severe side effects, prompting a re-evaluation of standard regimen. Past studies have demonstrated promise for Low-Dose Metronomic/LDM chemotherapy, defined as the frequent administration of low doses of chemotherapeutic drugs with no prolonged drug-free breaks. Yet relative activities of LDM taxanes and combinations with known anti-neoplasic agents have to be investigated. Methods: PC3, Du145 and LNCaP-derivative CL1cell lines were used to compare the effect of increasing doses of taxanes on cell proliferation, cell cycle distribution and apoptosis by crystal violet, propidium iodide and AnnexinV stainings, respectively. Autophagy was assessed by western blotting against Beclin1. In vivotumor growth was measured using CL1 control and CL1-PEDF xenografts. Phagocytosis was determined by cytotoxicity assay in CL1-macrophages co-cultures. Results: Our data showed that cabazitaxel/cbz-treated cells had a significantly lower EC50 compared to docetaxel/doc, with Du145 cells presenting the greatest differences. Both low-dose taxanes increased the sub-G0 cells population. However, the sub-G0 increase was significantly greater in cbz- than doc-treated Du145 cells, but not in PC3 and CL1. Accordingly, plasma membrane Annexin V elevation occurred in Du145 cells at lower doses of cbz than doc validating a higher efficacy due to increased apoptosis. Although Beclin1 levels remain unchanged in PC3 and CL1 cells, it was found up-regulated for all doses of cbz in Du145. In vivo, LDM cbz was significantly more efficient in curbing tumor growth than doc. This effect was markedly increased when cbz was combined with the angio-inhibitor and anti-tumor Pigment Epithelium-Derived Factor (PEDF); an effect that could be explained by increased phagocytosis. Conclusions: Our data demonstrate a higher efficacy of cbz on CRPC both in vitro and in vivo, and suggest that LDM taxane chemotherapy/PEDF combination could be used as a novel therapeutic strategy for CRPC.

In breast cancers, estrogen receptor (ER) levels are highly correlated with response to endocrine therapies. We sought to define mechanisms of estrogen (E) signaling in a solid breast tumor model using gene expression profiling. ER+ T47D-Y human breast cancer cells were grown as xenografts in ovariectomized nude mice under four conditions: 1) 17β-estradiol for 8 wk (E); 2) without E for 8 wk (control); 3) E for 7 wk followed by 1 wk of E withdrawal (Ewd); or 4) E for 8 wk plus tamoxifen for the last week. E-regulated genes were defined as those that differed significantly between control and E and/or between E and Ewd or control and Ewd. These protocols generated 188 in vivo E-regulated genes that showed two major patterns of regulation. Approximately 46% returned to basal states after Ewd (class I genes); 53% did not (class II genes). In addition, more than 70% of class II-regulated genes also failed to reverse in response to tamoxifen. These genes may be interesting for the study of hormone-resistance issues. A subset of in vivo E-regulated genes appears on lists of clinical ER discriminator genes. These may be useful therapeutic targets or markers of E activity. Comparison of in vivo E-regulated genes with those regulated in identical cells in vitro after 6 and 24 h of E treatment demonstrate only 11% overlap. This indicates the extent to which gene expression profiles are uniquely dependent on hormone-treatment times and the cellular microenvironment.

Abstract Biomarker-based chemotherapy with increased efficacy and prolonged disease-free survival are urgently needed in pancreatic cancer (PDAC). A 15-20% subset of PDAC tumors carry mutations in DNA repair pathway (BRCA1/BRCA2/PALB2/RAD51/ATM/FANCD2). Additionally, mutations in nucleotide excision repair (NER) genes (ERCC2/3/4/5/6) have been reported in ~5% of PDAC. LP184 is a novel synthetic small molecule acylfulvene analog. Using precise synthetic chemistry, we determined that only a negative enantiomer of LP184 is converted to an active alkylating agent in the strict dependency on oxidoreductase, prostaglandin reductase 1 (PTGR1). By computational analyses, we demonstrate a strong positive correlation of LP184 sensitivity with PTGR1 transcript levels (r=0.89, p<10−15) in a broad panel of cancer cell lines. Once activated by PTGR1, highly reactive LP-184 nucleophile creates covalent DNA adducts that are selectively repaired via Nucleotide Excision Repair (NER) mechanism coupled to transcription (TC-NER) and/or homologous recombination (HR). We reasoned that mutation or expression driven TC-NER and HR deficiency would predispose PDAC cells to increased sensitivity to LP184. To test the idea of LP184 activity in DNA repair-deficient tumors, we evaluated LP184 chemosensitivity in genetically defined PDAC models in vitro, ex vivo, and in xenografts. Testing in six different pancreatic cancer cell lines (Capan-1, CFPAC-1, Panc1, MiaPaCa2, Panc03.27 and BxPC-3) resulted in very potent inhibition with LP184 IC50 values ranging from 114 to 182 nM. In this cell line panel, LP184 sensitivity correlated negatively with transcript levels of an NER pathway gene ERCC8 (r = -0.94). In comparison to these PDAC cell lines, a normal pancreatic epithelial cell line HPNE was 3-6 times less sensitive to LP184 (IC50 670 nM). Ex vivo cultures of 4 out of 5 low-passage patient-derived xenografts with HR deficiency showed nanomolar sensitivity to LP184 with IC50s ranging from 45 to 270 nM. These tumor graft models which were at least 6 times less sensitive to olaparib in the same assay. Depletion of ERCC4 enhanced sensitivity to LP184 about 2-fold relative to the parental cell line. To define PTGR1 as a biomarker for LP184 activity, we used CRISPR/Cas9-mediated gene editing to deplete PTGR1 expression. We found PTGR1-null Capan-1 cell line-derived xenografts were poorly sensitive to LP184, whereas PTGR1-expressing xenografts showed near complete tumor regression in all LP184 treated animals with 109% tumor growth inhibition relative to the control group in this study. Furthermore, PTGR1 depleted cells were completely resistant to LP184 in vitro. Our preclinical data demonstrate that PDAC models carrying a range of DNA repair pathway mutations are highly sensitive to LP-184 in vitro and in vivo. Increased PTGR1 expression is a validated biomarker for LP184 cytotoxicity, and is the exclusive convertase of LP184 to an active alkylator drug. We anticipate LP184 will extend the therapeutic opportunities to a large subset of PDAC patients carrying these genetic alterations. Citation Format: Diana Restifo, Aditya Kulkarni, Caleb Schimke, Joseph McDermott, Umesh Kathad, Kishor Bhatia, Panna Sharma, Igor Astsaturov. LP184, a novel alkylating agent, is highly effective in pancreatic cancers with DNA damage repair defects [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-036.

Pancreatic cancer (PC) is anticipated to be second only to lung cancer as the leading cause of cancer-related deaths in the United States by 2030. Surgery remains the only potentially curative treatment for patients with pancreatic ductal adenocarcinoma (PDAC), the most common form of PC. Multiple recent preclinical studies focus on identifying effective treatments for PDAC, but the models available for these studies often fail to reproduce the heterogeneity of this tumor type. Data generated with such models are of unknown clinical relevance. Patient-derived xenograft (PDX) models offer several advantages over human cell line-based in vitro and in vivo models and models of non-human origin. PDX models retain genetic characteristics of the human tumor specimens from which they were derived, have intact stromal components, and are more predictive of patient response than traditional models. This review briefly describes the advantages and disadvantages of 2D cultures, organoids and genetically engineered mouse (GEM) models of PDAC, and focuses on the applications, characteristics, advantages, limitations, and the future potential of PDX models for improving the management of PDAC.

Purpose: Compare pancreatic ductal adenocarcinoma (PDAC), preclinical models, by their transcriptome and drug response landscapes to evaluate their complementarity. Experimental Design: Three paired PDAC preclinical models—patient-derived xenografts (PDX), xenograft-derived pancreatic organoids (XDPO) and xenograft-derived primary cell cultures (XDPCC)—were derived from 20 patients and analyzed at the transcriptomic and chemosensitivity level. Transcriptomic characterization was performed using the basal-like/classical subtyping and the PDAC molecular gradient (PAMG). Chemosensitivity for gemcitabine, irinotecan, 5-fluorouracil and oxaliplatin was established and the associated biological pathways were determined using independent component analysis (ICA) on the transcriptome of each model. The selection criteria used to identify the different components was the chemosensitivity score (CSS) found for each drug in each model. Results: PDX was the most dispersed model whereas XDPO and XDPCC were mainly classical and basal-like, respectively. Chemosensitivity scoring determines that PDX and XDPO display a positive correlation for three out of four drugs tested, whereas PDX and XDPCC did not correlate. No match was observed for each tumor chemosensitivity in the different models. Finally, pathway analysis shows a significant association between PDX and XDPO for the chemosensitivity-associated pathways and PDX and XDPCC for the chemoresistance-associated pathways. Conclusions: Each PDAC preclinical model possesses a unique basal-like/classical transcriptomic phenotype that strongly influences their global chemosensitivity. Each preclinical model is imperfect but complementary, suggesting that a more representative approach of the clinical reality could be obtained by combining them. Translational Relevance: The identification of molecular signatures that underpin drug sensitivity to chemotherapy in PDAC remains clinically challenging. Importantly, the vast majority of studies using preclinical in vivo and in vitro models fail when transferred to patients in a clinical setting despite initially promising results. This study presents for the first time a comparison between three preclinical models directly derived from the same patients. We show that their applicability to preclinical studies should be considered with a complementary focus, avoiding tumor-based direct extrapolations, which might generate misleading conclusions and consequently the overlook of clinically relevant features.

Abstract Patient-derived model systems serve as a platform for translational research representing the heterogeneity of human cancers, and their success in recapitulating disease-driving genomic alterations is well-documented. While recent studies have demonstrated genomic and functional divergence in patient-derived models with passaging, the need for accurate preclinical models remains. Glioblastoma (GBM) is the most common and aggressive primary brain tumor, and thus far preclinical models have failed to consistently replicate the responses found in patients. We therefore aimed to evaluate the multi-omic fidelity of low-passage GBM model systems across in vitro and in vivo environments and to elucidate the molecular features in which they differ. To this end we established a biobank of glioma direct-from-patient orthotopic xenograft (GliomaPDOX) models and primary gliomasphere cultures (GSCs) and performed whole-exome and RNA sequencing of over 40 purified patient tumors and their matched GliomaPDOXs and GSCs to facilitate paired comparisons across a gradient of full tumor microenvironment (TME) presence. We observed global genomic and transcriptomic fidelity in both systems, but specific programmatic gene expression differences associated with cell-cell interactions in the brain TME, glial cell identity, and in vitro GSC-forming ability. GSCs and GSC-forming ability are strongly associated with an astrocytic gene expression signature, while more stem-like and oligodendrocytic patient tumors including IDH- and H3F3A-mutant GBMs more successfully engraft in GliomaPDOXs. This result implicates the brain TME as a support system for these more stem/oligo-like tumors. Transcription factor network analysis identified regulators of the NOTCH and MYC pathways as strongly enriched in this subgroup of patient tumors and their derivative xenografts, and provides potential targets for therapeutic intervention in near future experiments. Collectively, these findings underline the critical role of the TME in defining GBM cell state, reveal the heterogeneity of TME dependence across patient tumors, and link this dependency to therapeutically actionable molecular features.

Object Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. Methods Primary cells from a sacral chordoma, called “DVC-4,” were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγnull mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0–3, heterogeneity scores of 0–1) were reported and evaluated to test differences across groups. Results The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2–3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2–3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1–2) with a predominantly heterogeneous staining pattern. Conclusions This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.

Abstract Background: Acute myeloid leukemia (AML) has very poor long-term survival with traditional therapies. AML has a diverse pathogenesis and likely represents multiple different diseases. Various epigenetic effector proteins are altered in AML by mutation, over-expression, or compartmental displacement and these changes maintain transcriptional programs important for leukemogenesis. The bromodomain and extra-terminal domain (BET) proteins, including BRD2, BRD3 and BRD4, play roles in many cellular functions important to leukemogenesis, such as super-enhancer function, transcriptional elongation, histone acetylation and cell cycle progression. In particular, AML cells depend on BRD4 for expression of the pro-survival proteins MYC and BCL2. BRD4 has therefore become an attractive target for novel therapeutics. PLX51107 is a novel BET inhibitor with a unique binding mode in the acetylated lysine binding pocket of BRD4 that differentiates it from other compounds under investigation. Our group has previously shown this compound to have antineoplastic activity in models of aggressive B cell malignancies. We have now investigated the anti-leukemic properties of PLX51107 in both in vitro and in vivo models of AML. Results: PLX51107 treatment potently reduced viability and proliferation of the human AML cell lines MV4-11, MOLM-13, OCI-AML3, and Kasumi-1, with IC50 of 0.17, 1.8, 0.2 and 0.2 μM, respectively. We then evaluated the in vitro activity of PLX51007 in primary human AML samples. PLX51107 inhibited the proliferation of primary human AML cells co-cultured with HS5 stromal cells. For nearly all samples tested (n=9), the IC50 of PLX51007 was less than 1 μM (average = 0.41 μM, range 0.039 - 1.5 μM). Notably, PLX51107 showed efficacy across a broad range of AML risk groups, including samples with adverse risk features such as 11q23 abnormalities and FLT3-ITD mutations. In comparison, for the same AML samples, the average IC50 for JQ1 was 0.71 μM (range 0.02 - 3.3 μM) and for cytarabine was 3.5 μM (range 0.33 to >10 μM). Furthermore, PLX51107 treatment reduced the clonogenicity of primary AML cells. Following incubation of AML cells in 1 μM PLX51107, there was significantly decreased colony formation (p<0.05) in drug-free, cytokine-supplemented methylcellulose media. We next examined the efficacy of PLX51107 in vivo, utilizing luciferase labeled MV4-11 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 47 days) compared to vehicle treated control animals (median 30 days, p< 0.001). Weekly measurement of bioluminescence showed decreased disease burden in PLX51107 treated mice. In addition, human peripheral blood CD45 / CD33 double positive cells were significantly decreased in treated animals. Histologic analysis conducted at day 16 showed decreased leukemic burden in the bone marrow of the PLX51107 treated animals. In addition, examination of tissues from moribund mice at time of euthanasia demonstrated fewer leukemia cells in the spleen, liver and bone marrow. Conclusions: Collectively, our results show pre-clinical activity of PLX51107 in AML, supporting further development of this compound in clinical trials for relapsed or refractory myeloid malignancies. We are currently working to define downstream targets of PLX51107 action and developing patient derived AML xenografts to further characterize the in vivo effects of PLX51107. Disclosures Walker: Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding.

Abstract Diffuse invasion is one of the key features that make GBM particularly difficult to treat. We hypothesize that direct comparison of matched invasive (GBMINV) and tumor core GBM cells (GBMTC) would facilitate the discovery of drivers of pediatric GBM (pGBM) invasion. However, GBMINV cells are extremely difficult to obtain from normal brain tissues because aggressive surgical resection of normal tissue carries the risk of serious neurological deficits. Most past and current studies on GBM invasion were and are forced to utilize the resected primary tumor masses. To overcome this barrier, we utilized a panel of 6 pediatric patient tumor-derived orthotopic xenograft (PDOX) mouse models to isolate matching pairs of GBMTC cells and GBMINV cells and confirmed a significantly elevated invasive capacity in GBMINV cells both in vitro and in vivo. Global profiling of 768 human microRNA using a real-time PCR-based Taqman system identified 23 microRNAs were upregulated in the GBMINV cells in at least 4 of the 6 pGBM models as compared with the matching GBMTC cells. We subsequently showed that silencing the top three miRNAINV, miR-126, miR-369-5p, and miR-487b, suppressed tumor cell migration in vitro (both as neurospheres and monolayer cultures) without affecting cell proliferation, and blocked pGBM invasion in mouse brains. Integrated analysis of the mRNA profiling of the same set of GBMTC and GBMINV cells revealed the affected signaling pathways and identified KCNA1 as the sole common computational target gene of the three miRNAINV. Treatment of three pairs of GBMTC and GBMINV cells with two KCNA1 inhibitors, ADWX1 and Agitoxin 2, caused significant suppression of pGBM cell migration in vitro. In conclusion, this study revealed an intrinsically elevated invasive phenotype in GBMINV cells, identified miR-126, -369-5p, and -487b as novel drivers of pGBM invasion, and characterized KCNA1 as a potential therapeutic target for arresting pGBM invasion.