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  • American Association for Cancer Research (AACR)  (24)
  • 1
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    Online Resource
    Abstract 4275: Production and characterization of ErbB1 (EGFR) associated with nanolipoprotein particles via cell-based and cell-free methods.
    American Association for Cancer Research (AACR) ; 2013
    In:  Cancer Research Vol. 73, No. 8_Supplement ( 2013-04-15), p. 4275-4275
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 73, No. 8_Supplement ( 2013-04-15), p. 4275-4275
    Abstract: The four members of the ErbB family of mammalian receptor tyrosine kinases are essential for the development and maintenance of a variety of tissues. Aberrant activation or over-expression of these receptors leads to the progression of epithelial and brain tumors. An understanding of the mechanistic and structural changes that occur to activate the intracellular receptor tyrosine kinase domain via extracellular ligand binding and receptor dimerization is desirable for identifying specific and efficient methods to inhibit signal transduction. Previous methods to study this process achieved low success due to low protein yield, poor water solubility of the membrane protein, and the large size of receptor. We report progress on overcoming these technical hurdles by forming the ErbB family members in nanolipoprotein particles (NLPs), which are ∼20 nm disc-shaped cell membrane analogs. Here we compare an optimized cell-based method with a novel cell-free method to produce NLPs containing the ErbB1 receptor, also called EGFR. The cell-produced ErbB1-NLPs retain kinase activity compared to cell-free produced material with higher yields. However, the cell-free produced receptors have regions of correct folding and can be produced in less than a day. The cell-free produced constructs can be labeled conveniently with unnatural amino acids to enable characterization of dimerization interactions. These two receptor production systems can be utilized synergistically to enable new studies of conformational changes associated with ligand binding or protein mutations, as well as to identify small molecule inhibitors of these receptors. Citation Format: Tiffany M. Scharadin, Christina Takanishi, Wei He, Matthew Saldano, Kermit L. Carraway, Matthew A. Coleman, Paul T. Henderson. Production and characterization of ErbB1 (EGFR) associated with nanolipoprotein particles via cell-based and cell-free methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4275. doi:10.1158/1538-7445.AM2013-4275
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2013-4275
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2013
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  • 2
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    Abstract 3321: Using NLPs to study EGFR structure, activation, and inhibition
    American Association for Cancer Research (AACR) ; 2014
    In:  Cancer Research Vol. 74, No. 19_Supplement ( 2014-10-01), p. 3321-3321
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 74, No. 19_Supplement ( 2014-10-01), p. 3321-3321
    Abstract: The mammalian ErbB receptor tyrosine kinase family is critical for the development and maintenance of a variety of tissues. Though not completely understood, the mechanism of ErbB receptor activation involves the binding of ligand to the extracellular domain leading to a conformational change that allows dimerization and phosphorylation to initiate downstream signaling pathways. Mutations, amplification, and aberrant activation of these receptors lead to oncogenesis and tumor progression in several cancer types, including lung, breast, and colon. Current ErbB-targeted therapies include monoclonal antibodies and tyrosine kinase inhibitors. These treatments are initially effective but many tumors develop resistance, necessitating the discovery of a more specific and efficient drug. Studying the ErbB receptors is often difficult because of their large size and poor water solubility. Here we report success in assembling EGFR into nanolipoprotein particles (NLPs) to study activation and inhibition of the correctly-folded, full-length, and active receptor. NLPs are ∼20 nm cell membrane analogs composed of an apolipoprotein surrounding a lipid bilayer. We produced a homogenous population of EGFR-NLPs, by FLAG-purification of EGFR from mammalian cells, which are of the correct size, phosphorylated, and can be quantified. Furthermore, these EGFR-NLPs can be utilized as a novel target in a one-bead-one-compound (OBOC) screen of small molecules and peptides to identify unique therapeutics. Studies of ligand binding, kinase activity, and EGFR structure are ongoing. Future directions are to incorporate disease-relevant EGFR mutations into NLPs. The T790M mutation is of particular interest because it is a treatment-induced mutation observed in half of all non small cell lung cancers and confers resistance to current ErbB-targeted therapies. Citation Format: Tiffany M. Scharadin, Matthew Saldana, Michael Schlein, Steven Hoang-Phou, Denise Trans, Dennis Chang, Wei He, Kit Lam, Kermit L. Carraway, Matthew A. Coleman, Paul T. Henderson. Using NLPs to study EGFR structure, activation, and inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3321. doi:10.1158/1538-7445.AM2014-3321
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2014-3321
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2014
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  • 3
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    Abstract 3333: In vitro-based biochemical studies of erbb2 gene variation to address racial disparities in breast cancer mortality
    American Association for Cancer Research (AACR) ; 2014
    In:  Cancer Research Vol. 74, No. 19_Supplement ( 2014-10-01), p. 3333-3333
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 74, No. 19_Supplement ( 2014-10-01), p. 3333-3333
    Abstract: According to a 2012 study by the U.S. Center for Disease Control and Prevention (CDC), African-American women had a 41% higher rate of breast cancer mortality during 2005-2009 than did Caucasian women. Although overall cancer mortality rates have declined significantly in the U.S. since the early 1990s, the black-white mortality gap has been steadily growing. The underlying causes of this racial disparity are likely diverse, and include factors such as lower quality of care and reduced access to mammographic screening. However, more aggressive tumor biology may also contribute significantly to racial disparity in breast cancer outcomes. Recently, a low frequency single nucleotide polymorphism (SNP) of the HER2 receptor tyrosine kinase, W452C, which occurs predominantly in African-American women (about 10% frequency), was significantly correlated with breast cancer. This study reported that tumors from patients harboring W452C do not amplify the erbB2 (HER2) gene or overexpress the protein, suggesting that this variant may contribute to breast cancer development through a mechanism distinct from the amplification common to HER2-positive breast cancer patients. Understanding the structural and functional differences associated with W452C will allow us to identify new strategies for customized breast cancer treatments for African American women. To address the role of the W452C SNP in breast cancer we applied a cell-free in vitro reconstitution system that uses nanolipoprotein particles (NLPs) to solubilize and support functional membrane proteins. Cell-free produced wild type HER2 and W452C were tested for tyrosine phosphorylation as well as specific binding to therapeutic anti-HER2 monoclonal antibodies trastuzumab and pertuzumab. Our results showed comparable tyrosine phosphorylation levels for both wild type and W452C HER2, suggesting that the variant itself might not alone be a driver of cancer. We observed a decreased binding affinity of trastuzumab for W452C compared to wild type HER2, suggesting that W452-positive patients might not respond to trastuzumab treatment. On the other hand, we found that W452C has a higher affinity for pertuzumab. Overall, our studies suggest that the W452C variant may be differentially sensitive to clinically pertinent therapeutic agents. Further characterization of this variant will be important for the development of more effective and precise therapeutic interventions. This in turn could lead to targeted measures that help address racial disparity problems in breast cancer mortality. Citation Format: Wei He, Matthew Saldana, Tiffany Scharadin, Steven Hoang-Phou, Denise Trans, Dennis Chang, Kermit Carraway, Paul Henderson, Matthew A. Coleman. In vitro-based biochemical studies of erbb2 gene variation to address racial disparities in breast cancer mortality. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2014-3333
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2014-3333
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2014
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  • 4
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    Abstract 2741: Loss of Cadherin-11 in PDAC induces altered immune cell infiltration and remodels stromal landscape
    American Association for Cancer Research (AACR) ; 2021
    In:  Cancer Research Vol. 81, No. 13_Supplement ( 2021-07-01), p. 2741-2741
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 81, No. 13_Supplement ( 2021-07-01), p. 2741-2741
    Abstract: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer with very few treatment options. Less than 10 percent of patients diagnosed with PDAC survive 5 years post diagnosis. Mutations in CDKN2A, SMAD4, KRAS and P53 have been well linked to the development of PDAC. Preclinical murine models have been developed that leverage key driver mutations and have significantly contributed to our understanding of PDAC. One such genetically engineered mouse model (GEMM) that has emerged as an important tool in PDAC investigations is the KPC mouse (LSL-KrasG12D/+LSL-Trp53R172H/+Pdx-1-Cre) that spontaneously develops pancreatic tumors at ~14-16 weeks of age. Cadherin-11 (Cdh11), a cell-to-cell adhesion molecule, is highly expressed in desmoplastic stroma, a characteristic of PDAC, that leads to difficulties in drug accessibility and has been hypothesized to contribute to chemotherapeutic resistance. However, the mechanisms by which Cdh11 deficiency in the stromal microenvironment of PDAC-bearing mice (KPC) influences therapeutic outcomes, has yet to be fully understood. Single-cell RNA sequencing (scRNAseq) of both the non-immune (CD45-) and immune (CD45+) cellular compartments of tumor bearing (KPC/Cdh11+/+), tumor bearing Cdh11 deficient (KPC/Cdh11+/-), non-tumor bearing Cdh11 deficient (Cdh11-/+) and wildtype (KP) were performed. We observed changes in the abundance and types of infiltrating immune cells (T-cells, B-cells, myeloid lineage cells) of KPC/Cdh11+/- tumors when compared to tumors harvested from KPC/Cdh11+/+ mice. KPC/Cdh11+/+ pancreata had significantly more myeloid cells while KPC/Cdh11+/- tumors favored an increase in the numbers of infiltrating B- and T- cells. Genes upregulated in infiltrating T-cells specific to KPC/Cdh11+/+ mice include Spp1, Ifi30, Apoe, Ifitm3, Fn1. The increase in B and T cell infiltration was specific to the Cdh11-/+ deficient background, since both pancreata from KPC/Cdh11+/- and Cdh11-/+ mice had elevated levels of infiltration, compared to the KPC group. We also observed a decrease in the number of antigen-presenting cancer associated fibroblasts (apCAFs) in Cdh11-/+ and KPC/Cdh11+/- pancreata, denoted by the lack of CD74+ fibroblasts. Further validation of these findings will help to define the role of Cdh11-/+ in modulating B and T-cell behavior in addition to providing insight into Cdh11-/+ as a therapeutic target for PDAC through altering the tumor microenvironment. Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas Hum, Ivana Peran, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela Loots. Loss of Cadherin-11 in PDAC induces altered immune cell infiltration and remodels stromal landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2741.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2021-2741
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2021
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  • 5
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    Abstract 2757: Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma
    American Association for Cancer Research (AACR) ; 2020
    In:  Cancer Research Vol. 80, No. 16_Supplement ( 2020-08-15), p. 2757-2757
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 80, No. 16_Supplement ( 2020-08-15), p. 2757-2757
    Abstract: Pancreatic ductal adenocarcinoma (PDAC) is one of the most incurable types of cancer. Cancer that develops in the acinar cells of the pancreas is typically not diagnosed until later stages, such as stage 3 or 4. As such, this form of cancer is particularly lethal with only 9% of patients reaching 5-year survival. PDAC is known to have a particularly dense extracellular matrix composed of fibroblasts, which have been previously shown to play an important role in promoting resistance to drug therapy. Characterization of the stromal networks involved in PDAC tumor development as well as protein markers of fibroblast subpopulations within the tumor stroma are critical to developing new fibroblast-targeted therapeutic approaches as well as understanding key signaling molecules that ultimately promote tumor progression and drug resistance. Single-cell RNA sequencing (scRNAseq) was utilized to analyze cancer-associated fibroblasts (CAFs) from KPC mouse-derived, MT3 subcutaneous murine allografts along with two fibroblast lines derived from human PDAC tumors: CRC-811 and IA-1340. Single cell gene expression profiling and subsequent analysis of MT3-derived CAFs resulted in the identification of three CAF subpopulations including a myofibroblast subpopulation that expressed high levels of smooth muscle actin (Acta2), which was also observed in both human samples. Fibroblast subpopulations enriched in Acta2 expression, expressed high levels of Wnt5a along with several other secreted factors including Tgfb1, Tgfb2 and Ctgf. Wnt5a is a secreted protein that activates non-canonical Wnt signaling pathways and is known to regulate normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However Wnt5a is not natively expressed in MT3 cancer cells derived from syngeneic tumors, but potentially in the stroma. We hypothesize it is exclusively derived from fibroblasts. Previously it has been shown that Wnt5a inhibition suppressed gastric cancer metastasis, therefore further validation of the role of myofibroblast-derived Wnt5a on PDAC disease progression is warranted. This study received funding by LDRD 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM Release Number: LLNL-ABS-798442. Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2757.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2020-2757
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2020
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  • 6
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    Abstract 2810: Epithelial-mesenchymal hybrid population changes from monolayer, spheroid, and tumoroid ex vivo culture of syngeneic murine mammary tumors
    American Association for Cancer Research (AACR) ; 2020
    In:  Cancer Research Vol. 80, No. 16_Supplement ( 2020-08-15), p. 2810-2810
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 80, No. 16_Supplement ( 2020-08-15), p. 2810-2810
    Abstract: Epithelial to mesenchymal transition (EMT) of cancer cells during tumor progression has been implicated in tumor initiation, growth, invasion, metastasis, colonization and resistance to therapy. In building a successful in vitro tumor model, it is critical to recapitulate in vivo cancer cell heterogeneity inclusive of both cell types and transition state present. This study investigates the EMT hybrid states of mouse triple negative breast cancer cells during ex vivo culturing across multiple methodologies. Mouse mammary carcinoma (4T1) tumors were harvested from syngeneic, orthotopic mice and subsequently cultured using various methods over the course of 12 days. Ex vivo cultures were first assessed for retention of tumor cellular heterogeneity using endogenous Thy1.1 (CD90.1) expression via flow cytometry to distinguish 4T1 cancer cells from stromal derived cells. Cancer cell populations rapidly became the majority of cells in culture. After 3 days of ex vivo culture, we found the cancer cell population to have significantly expanded ~2-3 fold compared to the original tumor population, while other stromal cellular subtypes decreased. Additionally, monolayer culture of ex vivo cells contained significantly more cancer cells relative to the spheroid or tumoroid (tumor fragment) culturing techniques. Cancer cells from each culturing technique were also evaluated for loss of Epcam expression and mesenchymal cell fate in reference to the initial EMT distribution at time of isolation. 4T1 cells cast into hydrogel retained high proportions of cells undergoing EMT, evidenced by fewer cells expressing Epcam. However, all other in vitro conditions favored the expansion of cancer cells in an epithelial state compared to in vivo tumors. Subsequent analysis of EMT populations for transitional hybrid states based on CD51, CD61, and CD106 expression was conducted using flow cytometric analysis. We found that in vitro culturing promoted mesenchymal character and this selection was time dependent. Additionally, we found that spheroids cultured in hydrogel for 7 days most closely resembled early hybrid EMT cell states ratios found in vivo Future research aims to optimize ex vivo culturing methodologies to best retain cancer cell characteristics and behavior of tumors obtained from human biopsies. Alterations in growth conditions and identifying critical stromal populations of interest will be critical in development of optimized preclinical ex vivo tumor culture models for drug discovery or personalized treatment. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM: LLNL-ABS-798441 Citation Format: Nicholas R. Hum, Kelly A. Martin, Elizabeth Wheeler, Matthew A. Coleman, Gabriela G. Loots. Epithelial-mesenchymal hybrid population changes from monolayer, spheroid, and tumoroid ex vivo culture of syngeneic murine mammary tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2810.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2020-2810
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2020
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  • 7
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    Abstract PO065: Single cell transcriptomics of triple negative breast cancer allografts following chemotherapy treatment reveals increased T cell abundance in doxorubicin-sensitive tumors
    American Association for Cancer Research (AACR) ; 2021
    In:  Cancer Immunology Research Vol. 9, No. 2_Supplement ( 2021-02-01), p. PO065-PO065
    Details
    In: Cancer Immunology Research, American Association for Cancer Research (AACR), Vol. 9, No. 2_Supplement ( 2021-02-01), p. PO065-PO065
    Abstract: The contribution of stromal cells on drug response in primary tumors remains unclear. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile cells present in murine tumors with or without exposure to doxorubicin. Doxorubicin is a chemotherapeutic agent commonly used to treat TNBC by inhibiting cancer cell proliferation through intercalation of DNA and preventing topoisomerase II activity. In this study, murine TNBC 4T1 cell line was utilized to generate allograft tumors in immunocompetent BALB/c mice. Syngeneic 4T1 tumors exhibit a range of responsiveness to doxorubicin treatment. Tumor growth rates were monitored throughout the chemotherapeutic regiment then stratified into sensitive or resistant response. Cellular composition and behavior were then analyzed via single cell RNA sequencing (scRNA-seq) and flow cytometry 8 days post-doxorubicin chemotherapeutic administration mimicking clinical treatment. ScRNA-seq revealed decreases in tumor infiltrating lymphocytes after doxorubicin exposure. Furthermore, an increase in T cell abundance was discovered in tumors sensitive to doxorubicin treatment. This finding was further supported by flow cytometric analysis showing a tumor specific increase in all, CD4+, and CD8+ T cell populations relative to resistant tumors. Additionally, T-cell differentiation, exhaustion, and activation states were further examined from scRNA-seq data providing insights into functional properties of the tumor residing T cell populations undergoing chemotherapeutic treatment. Future work will focus on analyzing prognostic value of specific T-cell populations in disease regression following doxorubicin treatment. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas R. Hum, Aimy Sebastian, Sean F. Gilmore, David M. Gravano, Naiomy D. Rios-Arce, Kelly A. Martin, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Single cell transcriptomics of triple negative breast cancer allografts following chemotherapy treatment reveals increased T cell abundance in doxorubicin-sensitive tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO065.
    Type of Medium: Online Resource
    ISSN: 2326-6066 , 2326-6074
    URL: Article
    DOI: 10.1158/2326-6074.TUMIMM20-PO065
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2021
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  • 8
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    Abstract 130: Characterization of the tumor microenvironment using single cell transcriptomics of triple negative breast cancer allografts treated with doxorubicin
    American Association for Cancer Research (AACR) ; 2019
    In:  Cancer Research Vol. 79, No. 13_Supplement ( 2019-07-01), p. 130-130
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 79, No. 13_Supplement ( 2019-07-01), p. 130-130
    Abstract: It is currently unclear how stromal components affect drug response and the emergence of drug resistance, in primary tumors. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile individual cells present in murine tumors with or without exposure to doxorubicin (Dox). Dox is a common chemotherapeutic agent used to treat breast cancer which inhibits breast cancer proliferation by intercalating into DNA and preventing topoisomerase II activity. Several autonomous processes have been implicated in the development of chemoresistance yet the impact of stromal and immune cells on tumor progression is still poorly understood. In this study, TNBC 4T1 cell line were utilized to generate murine allograft tumors in immunocompetent BALB/c mice. Tumor composition was analyzed via single cell RNA sequencing after 3 and 7 days of doxorubicin chemotherapeutic regiment mimicking clinical treatment. Using Cell Ranger single cell software suite and Seurat R toolkit, single cell transcriptomic analysis identified the cellular composition of tumors through expression of cell-type specific genes. Stromal cell types such as endothelial, fibroblast and epithelial cells were assessed and quantified in the tumor microenvironment. Immune cell types including neutrophils, monocytes, macrophages, T-cells and B-cells were also identified in the stroma and the responses to doxorubicin treatment was determined based on the gene expression changes. In this study, cancer-associated fibroblasts and non-canonical tumor associated macrophage subpopulations are of particular interest. As expected, we found both qualitative and quantitative changes in specific subpopulations of stromal cells in response to Dox exposure. Identification of stromal and immune cell sub-types could also lead to improved diagnostic capabilities and tumor susceptibilities. Future studies modulating non-cancerous cells in the tumor microenvironment may increase efficacy of chemotherapeutics. Further elucidating the specific cellular subpopulations within the tumor microenvironment that shift in response to drug exposure may provide new therapeutic avenues. Understanding changes in cell populations within the drug exposed tumor microenvironment can aid in future drug development to specifically target cells least sensitive to chemotherapy exposure. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Sean Gilmore, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Characterization of the tumor microenvironment using single cell transcriptomics of triple negative breast cancer allografts treated with doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 130.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2019-130
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2019
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  • 9
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    Abstract 2528: Determining gene expression variability between in vitro and in vivo cancer models: Monolayer, spheroids, and mouse allografts
    American Association for Cancer Research (AACR) ; 2018
    In:  Cancer Research Vol. 78, No. 13_Supplement ( 2018-07-01), p. 2528-2528
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 78, No. 13_Supplement ( 2018-07-01), p. 2528-2528
    Abstract: An ideal pre-clinical environment that recapitulates in vivo growth conditions ex vivo is an essential pre-requisite for effective drug screening. Conventional monolayer culturing of cancer cells as a pre-clinical model have repeatedly failed to recapitulate responses seen clinically. While 3D culturing methods are able to generate some level of tumor heterogeneity including hypoxic core, cell-cell interactions, gradients of drug penetration, as well as cancer stem cell differentiation, these culturing methods fail to incorporate the complex heterogenous cell composition and transient fluxes in nutrients or drugs. To investigate the effects of culturing conditions on gene expression of cancer cells, RNA sequencing was performed on a mouse mammary carcinoma (4T1) cell line grown in a variety of culture conditions: 2D (monolayer) or 3D (spheroid). Additionally, gene expression analysis was performed on tumors derived from 4T1 cells injected subcutaneously (SQ) into the murine flank or orthotopically (OT) into the mammary fat pad of BALB/c mice. Pairwise analysis of RNA sequencing data identified 235 down- and 1029 up- regulated genes differentially expressed between the 2D and 3D culture methods. Differential expression identified genes involved in cell migration, extracellular matrix organization, cell adhesion, angiogenesis, hypoxic response, cell differentiation, as well as key cancer related pathways including TNF, Jak-STAT, and PI3K-AKt primarily upregulated in 3D culture. Similar differential expression was found for genes encoding extracellular matrix proteins, which may be reflective of the 3D environment. Both in vivo allograft models produced highly similar gene expression profiles with only 31 up- and 20 down- regulated genes differentially expressed between OT and SQ tumors. Down-regulated genes were enriched in transcriptional regulatory gene networks and up-regulated genes were enriched for signaling/secreted proteins. The gene expression profiles of in vivo tumors were significantly different when compared to the 2D (973 down regulated, 1971 upregulated genes); and 99 of these transcripts were only expressed in in vivo tumors, highlighting the increased heterogeneity of cell composition found in allografts. 3D culturing of cancer cells upregulates pathways known to be critical to tumor progression including genes known to be essential for adhesion, differentiation, and ECM remodeling however the gene expression profile of spheroids is significantly different than that of cancer cells expanded in vivo. Future culturing methods incorporating immune cells, cancer supporting cells such as fibroblasts and other stromal components are more likely to improve the phenotype of 3D cultured tumor cells. This study received funding from LLNL LDRD grant 17-ERD-121. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas R. Hum, Wei He, Aimy Sebastian, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Determining gene expression variability between in vitro and in vivo cancer models: Monolayer, spheroids, and mouse allografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2528.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2018-2528
    RVK:
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    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2018
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  • 10
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    Abstract 37: RNA-seq comparisons of in vitro and in vivo cancer model platforms: Monolayer, spheroids, immunodeficient, and syngeneic mouse model
    American Association for Cancer Research (AACR) ; 2019
    In:  Cancer Research Vol. 79, No. 13_Supplement ( 2019-07-01), p. 37-37
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    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 79, No. 13_Supplement ( 2019-07-01), p. 37-37
    Abstract: Preclinical cancer models have been vital contributors in minimizing this burden as well as understanding basic cancer cell biology, however conventional and modern cancer models do not accurately or reliably recapitulate the complex in vivo tumor environment. Clinical significance of discoveries made using in vitromodels requires an understanding of the limitations imparted from cancer cells in a non-native environment. An ideal pre-clinical cancer platform that mimicks in vivo molecular phenotypes is essential for achieving effective drug screening and personalized treatments. This study aims to elucidate biological processes deficient in conventional in vitro methods from in vivo grown allograft cancer cells via transcriptome analysis. The effects of culturing conditions on cancer cells were analyzed via whole transcriptome RNA sequencing on a mouse mammary carcinoma (4T1) cell line grown in multiple culture conditions: 2D (monolayer) or 3D (spheroid) constructs under static or dynamic flow in addition to 4T1 cells isolated from subcutaneous or orthotopically grown tumors into the mammary fat pad of immune-competent, BALB/c mice. Comparative analysis of whole transcriptomic profiles of 4T1 cells in differing culturing conditions reveals distinct biological processes fostered by their environment. Monolayer culture shows enrichment in gene ontologies promoting proliferation, cell cycle progression, and protein synthesis. Compared to monolayer culture all 3-dimensional culturing methods encouraged the expression of proteins known to be critical to tumor progression such as extracellular matrix remodeling, adhesion, and differentiation. Furthermore, spheroid culture introduced heterogeneity as evidenced by upregulation of hypoxic induced genes and regulation of multicellular organism development processes. As expected, 4T1 cells expanded in vivo upregulated genes associated with processes difficult to recapitulate in vitro such as cell migration, inflammatory response, and angiogenesis. 3D culturing methods are able to recapitulate aspects of tumor heterogeneity yet fail to incorporate the complex heterogeneous cell composition and transient fluxes in nutrients and drugs found in vivo. Findings from this study demonstrate the behavioral and transcriptional alterations imparted from environmental factors. Additionally, clinically relevant in vitro testing can be improved by the incorporation of factors found in the native tumor microenvironment to existing 3D culturing approaches. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Wei He, Monica L. Moya, William F. Hynes, Jonathan J. Adorno, Aubree Hinckley, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. RNA-seq comparisons of in vitro and in vivo cancer model platforms: Monolayer, spheroids, immunodeficient, and syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 37.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2019-37
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2019
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    detail.hit.zdb_id: 410466-3
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