Mouse Mammary Carcinoma Cell Line Research Articles

Abstract 5568: Understanding the role of immune exclusion in mammary tumor growth

Abstract Multiple solid tumor types, including triple-negative breast cancer (TNBC), respond poorly to immunotherapy due to low infiltration of CD8+ T cells into the tumor core. The extracellular matrix (ECM) and its components such as collagen have been found to contribute to immune cell exclusion. Discoidin domain receptor 1 (DDR1) is a collagen receptor known to be aberrantly expressed in TNBC among multiple solid tumor types. We have uncovered a previously unappreciated function of DDR1 in immune exclusion through shaping a collagen fiber-based tumor defense against anti-tumor immunity (Sun et al, Nature 599, 673-678 (2021)). Our study showed that deletion of DDR1 in mouse mammary carcinoma cell lines such as E0771 and M-Wnt (syngeneic with C57BL/6 strain background) promotes infiltration of CD8+ T cells and reduces tumor growth in an immunocompetent mouse model. Extending the published study to other immunocompetent mouse strain backgrounds, we observed that deletion of DDR1 in EMT6 cells, another immune excluded TNBC mouse model in the BALB/c strain, increased the infiltration of CD8+ T cells to the tumor core and reduced tumor growth. Data concerning the DDR1-dependent spatial architecture of the mammary tumor microenvironment will be presented. Identifying specific immune and stromal cell populations and proteins spatially modified in the presence or absence of DDR1 may aid in development of new stand-alone or combination anti-cancer treatments with anti DDR1 antibody. Citation Format: Payal Mitra, Jorge Azpurua, Huai-Chin Chiang, Rong Li. Understanding the role of immune exclusion in mammary tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5568.

Relatively Rare Populations of Invasive Cells Drive Progression of Heterogeneous Tumors.

Breast tumors often display an astonishing degree of spatial and temporal heterogeneity, which are associated with cancer progression, drug resistance, and relapse. Triple-negative breast cancer (TNBC) is a particularly aggressive and heterogeneous subtype for which targeted therapies are scarce. Consequently, patients with TNBC have a poorer overall prognosis compared to other breast cancer patients. Within heterogeneous tumors, individual clonal subpopulations may exhibit differences in their rates of growth and degrees of invasiveness. We hypothesized that such phenotypic heterogeneity at the single-cell level may accelerate tumor progression by enhancing the overall growth and invasion of the entire tumor. To test this hypothesis, we isolated and characterized clonal subpopulations with distinct morphologies and biomarker expression from the inherently heterogeneous 4T1 mouse mammary carcinoma cell line. We then leveraged a 3D microfluidic tumor model to reverse-engineer intratumoral heterogeneity and thus investigate how interactions between phenotypically distinct subpopulations affect tumor growth and invasion. We found that the growth and invasion of multiclonal tumors were largely dictated by the presence of cells with epithelial and mesenchymal traits, respectively. The latter accelerated overall tumor invasion, even when these cells comprised less than 1% of the initial population. Consistently, tumor progression was delayed by selectively targeting the mesenchymal subpopulation. This work reveals that highly invasive cells can dominate tumor phenotype and that specifically targeting these cells can slow the progression of heterogeneous tumors, which may help inform therapeutic approaches. The online version contains supplementary material available at 10.1007/s12195-023-00792-w.

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer.

As part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. Therefore, a better understanding of macrophage phenotype conversion in relevant models is needed to elucidate its potential roles in disease. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos following cytokine-mediated polarization and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each cancer cell line with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that iNos production is enhanced in these cancer models using PBI1, tracking increased fluorescence. Collectively, this work demonstrates that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.

Acquired radioresistance in EMT6 mouse mammary carcinoma cell line is mediated by CTLA-4 and PD-1 through JAK/STAT/PI3K pathway

Cancer recurrence is often associated with the acquisition of radioresistance by cancer tissues due to failure in radiotherapy. The underlying mechanism leading to the development of acquired radioresistance in the EMT6 mouse mammary carcinoma cell line and the potential pathway involved was investigated by comparing differential gene expressions between parental and acquired radioresistance cells. EMT6 cell line was exposed to 2 Gy/per cycle of gamma-ray and the survival fraction between EMT6-treated and parental cells was compared. EMT6RR_MJI (acquired radioresistance) cells was developed after 8 cycles of fractionated irradiation. The development of EMT6RR_MJI cells was confirmed with further irradiation at different doses of gamma-ray, and both the survival fraction and migration rates were measured. Higher survival fraction and migration rates were obtained in EMT6RR_MJI cells after exposure to 4 Gy and 8 Gy gamma-ray irradiations compared to their parental cells. Gene expression between EMT6RR_MJI and parental cells was compared, and 16 genes identified to possess more than tenfold changes were selected and validated using RT-PCR. Out of these genes, 5 were significantly up-regulated i.e., IL-6, PDL-1, AXL, GAS6 and APCDD1. Based on pathway analysis software, the development of acquired radioresistance in EMT6RR_MJI was hypothesized through JAK/STAT/PI3K pathway. Presently, CTLA-4 and PD-1 were determined to be associated with JAK/STAT/PI3K pathway, where both their expressions were significantly increased in EMT6RR_MJI compared to parental cells in the 1st, 4th and 8th cycle of radiation. As a conclusion, the current findings provided a mechanistic platform for the development of acquired radioresistance in EMT6RR_MJI through overexpression of CTLA-4 and PD-1, and novel knowledge on therapeutic targets for recurrent radioresistant cancers.

Abstract 6241: Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment

Abstract Although the established cancer cell lines exhibit less degrees of intratumoral heterogeneity when compared to human solid tumors, studies have suggested that, even within the same cancer cell line, substantial degree of heterogeneity may exist. In this study, we have performed single cell cloning experiment using 4T1 mouse mammary carcinoma cell line and compared the phenotypes of different clones. When compared to the parental 4T1 cell line, one clone (C12) showed markedly different phenotype by exhibiting less aggressive features in vitro and significantly slower tumor growth and metastasis in vivo. The tumor tissue of the C12 showed significant alteration in immune microenvironment as higher number of recruited immune cells including T and B cells. RNA sequencing of the C12 clones and parental 4T1 cells identified Macc1 as a significantly up-regulated gene in parental 4T1 cells. Metastasis-associated colon cancer 1 (MACC1) has been associated with poor prognosis and the promotion of metastasis within several types of cancer, especially colon cancer. The gene encoding the hepatocyte growth factor (HGF) receptor, MET, is a transcriptional target of MACC1. Next, we established Macc1-silenced 4T1 cells using the parental 4T1 cells and observed that Macc1-silencing resulted in a similar phenotype seen in the C12 clone; decreased cell proliferation and migration in vitro and slow tumor growth and metastasis in vivo. To mimic the metastasis in vitro, intravasation was determined as which tumor cells were co-cultured with HUVEC, human umbilical vein endothelial cells. We discovered tumor cell penetration into HUVEC cells was blocked in Macc1-silencing 4T1 group. According to CD45, CD3, and CD19 immunohistochemistry, we also defined immune cells, especially T and B cells, were highly infiltrated in Macc1-silencing tumor like as C12 clone tumor. These results were also reconstructed in EMT6, another TNBC mouse cell line model. In this study, we show that MACC1 can regulate tumor progression and metastasis by remodeling of immune microenvironment. Citation Format: Hye-Youn Son, Woo Hang Heo, Song Bin Li, Mingji Quan, Yu Jeong Her, Kyu-Jin Lee, Ju Hee Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6241.

Abstract PS3-07: Molecular imaging of hypoxia and granzyme B alterations during combination treatment with immunotherapy in triple negative breast cancer

Abstract Background: Although a portion of triple negative breast cancer (TNBC) is sensitive to chemotherapeutic treatment with agents such as paclitaxel (PTX), patients have a high risk of recurrence and short overall and progression-free survival. Clinical trials for TNBC using immune checkpoint inhibitors such as anti-programmed cell death 1 (PD1) have met some success; however, only a small percentage of patients have a positive response to immunotherapy and there is risk for severe side effects. Therefore, there is a need for improvements in monitoring and predicting patient-specific response. The goal of this study is to use non-invasive visualization of the tumor microenvironment to collect longitudinal information that can be used to study tumor response to immunotherapy. By applying positron emission tomography (PET) imaging techniques to investigate changes in the tumor microenvironment (hypoxia and immune cell activation), we seek to predict early response to immunotherapy and better identify which treatments will be effective for individual tumors in TNBC. Methods: A TNBC mouse mammary carcinoma cell line, 4T1, was transduced with CMV-luciferase and 2x105 cells were injected into the third mammary fat pad of 5-6 week old female Balb/c mice (N=28). Mice in cohort 1 received: PTX (10 mg/kg), anti-PD1 (200 µg), both, or vehicle control (saline) (n=4/group). Mice in cohort 2 received: anti-PD1 (n=5), combination PTX/anti-PD1 (n=4), or vehicle control (saline; n=3). Treatments were administered intraperitoneally on days 0, 2, and 5 for cohort 1 (n=16) who underwent granzyme B-PET imaging ([68Ga]-NOTA-GZP-PET) and on days 0, 2, 5, and 8 for cohort 2 (n=12) who underwent hypoxia imaging with [18F]-fluoromisonidazole (FMISO)-PET imaging. Bioluminescence (BLI) imaging and caliper measurements were performed to track tumor size changes at multiple timepoints and tumors were collected for histological validation on day 20. Mean standard uptake value (SUVmean) was calculated as percent of day 0, and statistical analyses were performed with unpaired t-tests and Wilcoxon-rank sum tests. Results: Voxel analysis of GZP-PET images revealed an 42.9% increase in T cell activation of TNBC tumors treated with single-agent PTX compared to anti-PD1 alone on day 3 (p=0.08). FMISO-PET revealed that tumors treated with anti-PD1 alone had lower hypoxic fraction compared to control group tumors (p=0.10) and tumors treated with combination PTX/anti-PD1 (p=0.17) on day 3. BLI data showed that treatment with PTX and anti-PD1 significantly decreased viability signal between days 3 and 6 for cohort 1 (p=0.04). Non-responders to treatment had a significantly higher tumor volume compared to responders starting on day 6 (p<0.05). SUVmean, indicating T cell activation, was significantly higher for responders compared to non-responders on days 3 and 6 (p<0.05). There was a signifciant decrease in hypoxia, as measured thorugh FMISO-PET SUVmean on day 6 for responders compared to non-responders (p=0.04). Conclusion: It is known that the tumor microenvironment can impact neovascularization, tumor cell invasion, tumor oxygenation and growth. Results from this study show that noninvasive PET imaging can provide data on changes in T cell activation and hypoxia in response to chemotherapeutic treatment and immunotherapy in TNBC. Utilizing clinically-translatable advanced imaging strategies to understand the biologically distinct features of the TNBC tumor microenvironment can aid in personalizing anti-cancer therapies. Acknowledgements: We thank the American Cancer Society for RSG-18-006-01-CCE, NIH NCI R01 CA240589, and NIH P30CA013148. Citation Format: Tiara S. Napier, Chanelle L. Hunter, Patrick N. Song, Ben M. Larimer, Anna G. Sorace. Molecular imaging of hypoxia and granzyme B alterations during combination treatment with immunotherapy in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-07.

Evaluation of photodynamic therapy and nuclear imaging potential of subphthalocyanine integrated TiO2 nanoparticles in mammary and cervical tumor cells

This study, subphthalocyanines (SubPc) and SubPc integrated TiO2nanoparticles (SubPc-TiO[Formula: see text] were synthesized as novel photosensitizers. Their PDT effects were evaluated. Furthermore, nuclear imaging potential of [Formula: see text]I-labelled SubPc/SubPc-TiO2were examined in mouse mammary carcinoma (EMT6) and cervix adenocarcinoma (HeLa) cell lines. The uptake results show that SubPc labelled with [Formula: see text]I radionuclide ([Formula: see text]I-SubPc) in EMT6 and HeLa cell lines was found to be approximately the same as in the WI38 cell line. However, the uptake values of SubPc-TiO2labelled with [Formula: see text]I ([Formula: see text]I-SubPc-TiO[Formula: see text] in EMT6 and HeLa cell lines were determined to be two times higher than in the WI38 cell line. In other words, the target/non-target tissue ratio was identified as two in the EMT6 and HeLa cell lines. [Formula: see text]I-SubPc-TiO2is promising for imaging or treatment of breast and cervix tumors. In vitro photodynamic therapy studies have shown that SubPc and SubPc-TiO2are suitable agents for PDT. In addition, SubPc-TiO2has higher phototoxicity than SubPc. As a future study, in vivo experiments will be held and performed in tumor-bearing nude mice.

Abstract 37: RNA-seq comparisons of in vitro and in vivo cancer model platforms: Monolayer, spheroids, immunodeficient, and syngeneic mouse model

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.

The effects of a CRISPR/Cas9 IL-6 knockout in 4T1 mammary carcinoma cells on myeloid-derived suppressor cells (MDSCs) and Th17/Th22 cells

Abstract The 4T1 mouse mammary carcinoma cell line is syngeneic to Balb/c mice and is an effective tumor model to study the interactions between the immune system and metastatic breast cancer. The pro-tumor effects that myeloid-derived suppressor cell (MDSC) populations have on CD8+ T cells and Tregs are well known, but comparably less attention has been directed at the relationship between MDSCs and Type III immune Th17/Th22 cells in breast cancer. MDSCs and Th17/Th22 cells have been linked in other inflammatory settings, so in this study, we investigated MDSC, Th17 and Th22 cell interactions in 4T1 tumors, and the role of tumor-derived IL-6 in these interactions. IL-6 plays a key role in the induction of MDSCs under pathological conditions, as well as fate determining in Th maturation. Thus, the removal of IL-6 from 4T1 tumors allowed us to better characterize the source of these interactions. Populations of Monocytic (M)-MDSCs (CD11b+Ly-6G−Ly-6Chi), Polymorphonuclear (PMN)-MDSCs (CD11b+Ly-6G+Ly-6Clow), Th17 (CD3+CD4+RORγt+IFN-γ−IL-17A+IL-22−) and Th22 (CD3+CD4+IFN-γ−RORγt−IL-17A−IL-22+) cells were identified in peripheral blood, spleen, tumor and bone marrow (BM) of mice bearing wild-type and IL-6 knockout 4T1 tumors. Finally, we isolated 4T1 induced MDSCs (CD11b+Gr-1+)and cocultured them with healthy donors to investigate Th17 and Th22 population expansion. Our findings provide further insight into the interactions among MDSCs, Th17 and Th22 cells in a murine cancer model, which may inform immunotherapy efficacy and outcomes in human breast cancer.

AS1411 Aptamer Conjugated Gold Nanoclusters as a Targeted Radiosensitizer for Megavoltage Radiation Therapy of 4T1 Breast Cancer Cells

Introduction: In the present study, AS1411 aptamer conjugated gold nanoclusters (GNCs) have been introduced as a targeted radiosensitizer for enhancing megavoltage radiation therapy efficacy. RT has identified as an effective therapeutic modality for many different types of solid tumors. However, equal radiation beams absorption by tumor and surrounding healthy tissues is still a great challenge in RT which is caused by attenuation coefficient factor similarity. In order to overcome this challenge and to increase the efficacy of radiation therapy, radiosensitizers has been recommended. Materials and Methods: GNCs with ultra-small gold core and bovine serum albumin shell (BSA) as a versatile Nano-platform were synthesized and conjugated to AS1411 aptamer (Apt-GNCs). Due to nucleolin overexpression in breast cancer cells and high affinity of the AS1411 aptamer to nucleolin, mouse mammary carcinoma cell line (4T1) was selected as malignant cells and murine fibroblast (L929) was used as a normal cell line. Results: Flow cytometry assessments reveal a significant increase of GNCs uptake by the cancer cells in the presence of the aptamer as the targeting agent. Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements demonstrate 4 times more Apt-GNCs uptake by 4T1 cells than the normal cells at concentration ratio of 1:40 (4µM Aptamer and 160 µM GNCs at 24h incubation). Conclusion: Combination of megavoltage radiation therapy and Apt-GNCs as radiosensitizer causes effective cancer cells killing and obtaining dose enhancement factor (DEF) about 2.7 in clonogenic survival assay.

Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases

BackgroundSyndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan normally expressed primarily by epithelia and plasma cells, is aberrantly induced in stromal fibroblasts of breast carcinomas. Stromal fibroblast-derived Sdc1 participates in paracrine growth stimulation of breast carcinoma cells and orchestrates stromal extracellular matrix fiber alignment, thereby creating a migration and invasion-permissive microenvironment. Here, we specifically tested the role of stromal Sdc1 in metastasis.MethodsThe metastatic potential of the aggressive mouse mammary carcinoma cell lines, 4T1 and E0776, was tested in wild-type and genetically Sdc1-deficient host animals. Metastatic lesions were characterized by immunohistochemical analysis.ResultsAfter orthotopic inoculation, the lung metastatic burden was reduced in Sdc1−/− animals by 97% and more than 99%, in BALB/cJ and C57BL/6 animals, respectively. The difference in metastatic efficiency was maintained when the tumor cells were injected into the tail vein, suggesting that host Sdc1 exerts its effect during later stages of the metastatic cascade. Co-localization studies identified Sdc1 expression in stromal fibroblasts within the metastatic microenvironment and in normal airway epithelial cells but not in other cells (endothelial cells, α-smooth muscle actin positive cells, leucocytes, macrophages). The Ki67 proliferation index and the rate of apoptosis of the metastatic tumor cells were diminished in Sdc1−/− vs. Sdc1+/+ animals, and leucocyte density was indistinguishable. Sdc1-mediated metastatic efficiency was abolished when the animals were housed at a thermoneutral ambient temperature of 31 °C, suggesting that the host Sdc1 effect on metastasis requires mild cold stress.ConclusionsIn summary, Sdc1 is induced in the lung microenvironment after mammary carcinoma cell dissemination and promotes outgrowth of metastases in a temperature-dependent manner.

Abstract 2528: Determining gene expression variability between in vitro and in vivo cancer models: Monolayer, spheroids, and mouse allografts

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.

Encapsulation of camptothecin into pegylated polyelectrolyte nanocarriers

Camptothecin is a natural compound with excellent antitumor activity over a wide spectrum of human and animal cancers. However, the direct formulation is limited by insolubility in water and other biocompatible solvents, low stability in physiologic conditions, rapid plasma clearance and high systemic toxicity. In this study, pegylated polyelectrolyte nanocapsules were utilized to prepare camptothecin nanocarriers. Nanosize droplets containing the camptothecin were encapsulated in polyelectrolyte multilayer shells formed by sequential adsorption of polyelectrolytes technique. The nanocapsules were pegylated for passive targeting through the adsorption of the pegylated polyelectrolyte. The biological effects of encapsulated camptothecin on two tumor cell lines: mouse colon carcinoma cell line CT26-CEA and the mouse mammary carcinoma cell line 4T1 were studied. Obtained results suggest, that encapsulated drug, as well as free one, showed similar activity on the tested cells.

Mechanical ventilation promotes lung metastasis in experimental 4T1 breast cancer lung-metastasized models.

Background/purposeThe aim of this study was to test the hypothesis that mechanical ventilation (MV) during cancer surgery induces lung stroma/tissue milieu changes, creating a favorable microenvironment for postoperative lung metastatic tumor establishment.Materials and methodsIn Protocol A, female BALB/c mice were divided into an MV group and a control (no MV) group, both of which were anesthetized and subjected to intravenous injection of green fluorescent protein (GFP)-labeled mouse mammary carcinoma cell line (4T1) cells. After 24 h, the lung tissue was removed and the number of GFP-labeled 4T1 cells was calculated. In Protocol B, the clinically relevant mouse model of spontaneous breast cancer lung metastasis was used with surgical resection of the primary tumor to investigate the MV event that dictates postoperative lung metastasis. Female BALB/c mice were inoculated in the mammary fat pad with 4T1 cells. After 14-d growth, mice were anesthetized and divided into an MV group and a control (no MV) group during surgical procedures (mastectomy). Metastatic tumor burden was assessed two weeks after mastectomy by both macroscopic metastatic nodule count, hematoxylin–eosin histology, immunohistochemistry for the macrophage marker (CD68), and epithelial cell adhesion molecule (EpCAM).ResultsMV was associated with a significant increase in the number of circulating breast tumor cells (GFP-labeled 4T1 cells) remaining in the microvasculature of the lung (P<0.01). Immunohistochemical results showed increased infiltration of CD68-positive macrophages within injured lung parenchyma and metastatic tumor as well as increased expression of EpCAM in metastatic nodules. Postoperative metastases were more prevalent in the mechanically ventilated mice group compared to the non-ventilated group (P<0.05).ConclusionMV-induced lung metastasis occurs by attracting circulating tumor cells to the site of the lung injury and by accelerating the proliferation of preexisting micro-metastases in the lung. These observations indicate that the metastasis-enhancing effect of MV should be considered in general anesthesia during cancer surgery.

Salvia officinalis Induces Apoptosis in Mammary Carcinoma Cells Through Alteration of Bax to Bcl-2 Ratio

Salvia officinalis L. (sage) is one of the herbal plants frequently used in traditional medicine. Anti carcinogenic property of sage extract and the fact that it does not show toxic effect on normal cells, make this extract a proper candidate for cancer therapy. In order to assess the molecular mechanism underlying sage anticancer properties, the effect of sage hydro alcoholic extract was investigated on mouse mammary carcinoma cell line (4T1). Cultured cells were treated with different concentrations (0–1000 μg/mL) of Salvia officinalis extract for 24 h. Decrease in cell viability and increase in chromatin condensation were observed in a dose-dependent manner following treatment with sage extract. Most cultured cells were in the early stages of apoptosis in 100 μg/mL and in the late stages of apoptosis in 500 μg/mL of the extract. While Bax expression was increased significantly at 500 μg/mL of sage extract, no significant change was observed in the levels of Bcl2 expression in different concentrations of the extract compared to non-treated control. Therefore, Salvia officinalis hydro alcoholic extract shows cytotoxic and proapoptotic properties on mouse mammary carcinoma cells. Moreover, it seems that the induction of apoptosis in those cells is through increased in the ratio of Bax/Bcl2 as an apoptotic index.

Abstract 963: Ovarian-dependent SSM2ucd mouse mammary carcinoma cells depend on Cyp3a11 epoxygenase activity for proliferation

Abstract Introduction: Silencing of the human cytochrome P450 (CYP) monooxygenase enzyme CYP3A4 in the ER+ MCF-7 tumor cell line inhibits tumor growth in the mammary fat pad, but the relative roles of tumor cell intrinsic CYPs vs. vascular/microenvironment CYPs remain to be determined and a syngeneic animal model is needed to answer this question. The SSM2ucd ovarian dependent mouse mammary carcinoma cell line forms a transplantable estrogen-receptor positive (ER+) mouse mammary tumor in which growth dependence on cell intrinsic CYPs may be tested in vitro and in vivo. The SSM2ucd cell line is derived from 129SvJ: homozygous Stat1-null female mice, which develop ER+ mammary tumors (1,2). Expression profiling of spontaneously arising mouse mammary carcinomas in a TP53 KO model revealed that the CYP3A4 ortholog Cyp3a11 is up-regulated in these tumors. Methods: Cyp3a11 bactosomes were used to assay for NADPH dependent biosynthesis of EETs from arachidonic acid using an LC-MS/MS method. siRNA silencing and CRISPR/Cas9 knock out of the Cyp3a11 exons 2 and 3 was performed and the cell lines were characterized for EET dependence using an MTT assay. The highly potent inhibitor and chemical probe of CYP epoxygenase activity, hexyl-benzyl-biguanide (HBB), was used to test dependence of SSM2ucd cells on CYP epoxygenase activity. Total cellular EETs in SSM2ucd cells exposed to HBB at the IC50were measured by the LC-MS/MS method. Results: Cyp3a11 was shown to have robust NADPH dependent epoxygenase activity, while SSM2ucd cells were EET dependent for growth. Silencing of Cyp3a11 was demonstrated to inhibit proliferation of the SSM2ucd cell line, which was partly abrogated by EETs. CRISPR/Cas9 knock out of the Cyp3a11 in the SSM2ucd cell line was performed and exon 2 and exon 3 deleted cell lines were derived. SSM2ucd Cyp3a11 knock out cell lines were more sensitive to EET-induced proliferation under serum free condition than control lines. HBB potently inhibits the SSM2ucd cell line in vitro (IC50=25 uM) and reduces total cellular EET levels in this cell line at IC50. These results suggest that we may see inhibitory activity of HBB in a syngeneic 129/SvJ tumor transplantation model. Conclusions: The Cyp3a11 gene can be silenced in the SSM2ucd cell line, without disrupting cell viability, allowing further study of the roles of EETs in mouse mammary carcinoma proliferation, survival and clonogenicity. The Cyp3a11 gene has been knocked out in the SSM2 ucd cell line, allowing in vitro and in vivo studies of the roles of Cyp3a11 in the growth of ovarian dependent mouse mammary carcinoma. 1. Breast Cancer Res. 2012 Jan 20;14(1):R16 2. PLoS One. 2015 Jun 15;10(6):e0129895] Citation Format: Zhijun Guo, Patrick McGrarrah, Ted Bebi, Ashley Mooneyham, Alejandra Osorio, Cesar Herrera, Victor Arrieta, Sebastian Mohar, Irwin Hernandez, Robert Klink, Robert Cardiff, David A. Potter. Ovarian-dependent SSM2ucd mouse mammary carcinoma cells depend on Cyp3a11 epoxygenase activity for proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 963. doi:10.1158/1538-7445.AM2017-963

Abstract 5905: Cancer associated fibroblasts promote tumor metastasis coexisting with cancer cells in blood circulation

Abstract Background: Cancer associated fibroblasts (CAFs) are activated fibroblasts and an important player in the tumor microenvironment. Their activity promotes cancer cell proliferation, migration, and invasion, and metastasis. The most prognostic factor in tumor progression is metastasis. Cancer metastasis is a multi-step process that tumor cells detach from primary site, survive in the bloodstream, and seed in target organ. Although previous studies have focused on the interaction between CAFs and cancer cells in primary tumor site, the roles of CAFs in blood circulation remain largely unknown. We investigated the effect of CAFs coexisting with cancer cells in bloodstream on tumor metastasis in vivo mouse model, and also examined the effect of CAFs in subcutaneous tumor. Methods: We used female BALB/c-nu/nu mice and BALB/c mice in the experiments. Cancer cells were mouse mammary carcinoma cell lines 4T1 transfected with luciferase, colon cancer cell lines Colon 26 transfected with luciferase. Fibroblast cell lines were mouse embryonic fibroblast MEF and NIH-3T3. In venous injection mouse model, we injected 1 × 106 cancer cells alone or the same number of cancer and fibroblast co-cultured together. When we injected cancer cells transfected with luciferase, the mice were subjected to in vivo imaging system (IVIS) and measured luminescence intensity of metastatic sites. We have also harvested lung and compared its weight and metastatic nodule under microscopy. Furthermore, in subcutaneous tumor metastatic mouse model, cancer cells alone or mixed with cancer cells and CAFs were subcutaneously inoculated into the mice. Results: In the group of mice injected with cancer cells and fibroblasts, luminescent intensity of each lungs were higher than cancer cell alone. Harvested lung weight and the number of metastatic nodules were also higher in the group with cancer cells and fibroblasts.In subcutaneous model, mice inoculated with cancer cells and fibroblast had much more metastatic sites than cancer cells alone. Conclusions: Our data indicate that CAFs promote tumor metastasis by stimulating cancer cells in blood circulation, as well as in primary tumor site.These findings suggest that CAFs in both bloodstream and primary site could be a promising therapeutic target. CAF-targeted therapy could reduce tumor metastasis and improve the prognosis of cancer patients. Citation Format: Hajime Kashima, Kazuhiro Noma, Hiroaki Sato, Yuki Katsura, Takuya Kato, Toshiaki Ohara, Hiroshi Tazawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Cancer associated fibroblasts promote tumor metastasis coexisting with cancer cells in blood circulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5905. doi:10.1158/1538-7445.AM2017-5905

Chemotherapy-induced tumor dormancy and relapse

Abstract Breast cancer mortality is due to distant recurrence of the disease as a result of awakening dormant tumor cells that were established by cancer therapies. The mechanisms through which dormant tumor cells are maintained or escape from dormancy, and relapse remain elusive. One such mechanism that may play a role in tumor dormancy maintenance or escape is autophagy; a process important in all cells for the removal of damaged proteins and organelles. Although autophagy is a mechanism of tumor suppression, it also confers stress tolerance that enables tumor cells to survive under adverse conditions. Here, we wanted to determine the role of autophagy in tumor dormancy and sensitivity of dormant tumor cells to immunotherapy, i.e., IFN-gamma treatment. A mouse mammary carcinoma (MMC) cell line was established from the neu over-expressing FVBN202 transgenic mouse. We also used Adriamycin (ADR) for the establishment of chemotherapy-induced tumor dormancy, and blocked autophagy by chloroquine (CQ). We demonstrated that a transient blockade of autophagy by CQ during ADR treatment prolonged tumor dormancy, in vitro, but not in vivo. Also, we determined that dormant tumor cells established by ADR or ADR+CQ were more sensitive to IFN-gamma induced apoptosis compared with non-dormant, proliferating tumor cells. These observations suggest that autophagy could participate, in part, in tumor dormancy without affecting the sensitivity of dormant cells to IFN-gamma treatment.

Glycol porphyrin derivatives and temoporfin elicit resistance to photodynamic therapy by different mechanisms

The development of drug resistance is a major problem which often occurs during anticancer chemotherapies. Photodynamic therapy (PDT) has been studied as an alternative treatment modality for drug-resistant tumors, however the question of resistance to PDT and potential cross-resistance with chemotherapy has yet to be fully answered. To investigate the mechanism of resistance to PDT, we developed an in vitro experimental model system in a mouse mammary carcinoma cell line 4T1. We used two ethylene glycol derivatives of tetraphenylporphyrin, and tetraphenylchlorin derivative, temoporfin, as photosensitizers (PS). PDT-resistant clones were obtained by exposure to a set concentration of PS followed by irradiation with increasing light doses. PDT resistance to soluble glycol porphyrins was mediated mainly by increased drug efflux through ABCB1 (P-glycoprotein) as we demonstrated by specific ABCB1 knockdown experiments, which in turn rescued the sensitivity of resistant cells to PDT. In contrast, resistance raised to temoporfin, which is generally more lipophilic than glycol porphyrins, elicited mechanism based on sequestration of the drug to lysosomes. The resistance that is acquired from a particular PS could be overcome by using a different PS, which is not susceptible to the same mechanism(s) of resistance. Elucidation of the underlying mechanisms in various types of resistance might facilitate improvements in PDT treatment design.

PH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells

This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6–2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL.