Breast Tumors In Mice Research Articles

Hematopoietic stem cell specific V-ATPase controls breast cancer progression and metastasis via cytotoxic T cells.

The interaction of recruited immune effector cells and cancer cells within tumor microenvironment (TME) shapes the fate of cancer progression and metastasis. Many cancers including breast cancer, express a specific vacuolar ATPase (a2V) on their cell surface which acidifies the extracellular milieu helping cancer cell proliferation and metastasis. To understand the role of immune cell-associated-a2V during breast tumor pathogenesis, we knocked-out a2V (KO) from the hematopoietic stem cells (HSC) and generated breast tumors in mice. The a2V-KO mice developed faster growing, larger, and metastatic breast tumors compared to control mice. Further investigation of the TME revealed a significant reduction in the presence of CD4+ and CD8+ T cells in the a2V-KO tumors. Targeted RNA-Seq of the cells of the TME demonstrated that pro-inflammatory cytokines, death receptors, death receptor ligands, and cytotoxic effectors were significantly down-regulated within the a2V-KO TME. Interestingly, analysis of immune cells in the blood, spleen, and thymus of the non-tumor bearing a2V-KO mice revealed a significant decrease in CD4+ and CD8+ T cell populations. For the first time, this study demonstrates that inhibition of V-ATPase expression in HSC leads to a decrease in CD4+ and CD8+ T cell populations and thus promotes breast tumor growth and metastasis.

Abstract A28: Tumor cell intrinsic BPTF inhibits NK cell activity and the abundance of natural cytotoxicity receptor co-ligands

Abstract Using syngeneic BALB/c mouse breast cancer models, we show that the chromatin remodeling subunit bromodomain PHD finger transcription factor (BPTF) suppresses natural killer (NK) cell antitumor activity in the tumor microenvironment (TME). In culture, BPTF suppresses direct natural cytotoxicity receptor (NCR) mediated NK cell cytolytic activity to mouse and human cancer cell lines, demonstrating conserved functions. Blocking mouse NCR1 in vivo rescues BPTF KD tumor weights, demonstrating its importance for the control of tumor growth. We discovered that BPTF occupies heparanase (Hpse) regulatory elements, activating its expression. Increased heparanase activity results in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans (HSPGs). Using gain and loss of function approaches we show that elevated heparanase levels suppress NK cell cytolytic activity to tumor cells in culture. These results suggest that BPTF activates heparanase expression, which in turn reduces cell surface HSPGs and NCR co-ligands, inhibiting NK cell activity. Furthermore, gene expression data from human breast cancer tumors shows that elevated BPTF expression correlates with reduced antitumor immune cell signatures, supporting conserved roles for BPTF in suppressing antitumor immunity. Conditional BPTF depletion in established mouse breast tumors enhances antitumor immunity, suggesting that inhibiting BPTF could provide a novel immunotherapy. Citation Format: Kimberly Mayes, Zeinab Elsayed, Aiman Alhazmi, Michael Waters, Suehyb Alkhatib, Mark Roberts, Carolyn Song, Kristen Peterson, Vivian Chan, Nikhil Ailaney, Pumoli Malapati, Tana Blevins, Berislav Lisnic, Catherine Dumur, Joseph Landry. Tumor cell intrinsic BPTF inhibits NK cell activity and the abundance of natural cytotoxicity receptor co-ligands [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A28.

Field template-based design and biological evaluation of new sphingosine kinase 1 inhibitors

PurposeSphingosine kinase 1 (SK1) is a protooncogenic enzyme expressed in many human tumours and is associated with chemoresistance and poor prognosis. It is a potent therapy target and its inhibition chemosensitises solid tumours. Despite recent advances in SK1 inhibitors synthesis and validation, their clinical safety and chemosensitising options are not well described. In this study, we have designed, synthesised and tested a new specific SK1 inhibitor with a low toxicity profile.MethodsField template molecular modelling was used for compound design. Lead compounds were tested in cell and mouse cancer models.ResultsField template analysis of three known SK1 inhibitors, SKI-178, 12aa and SK1-I, was performed and compound screening identified six potential new SK1 inhibitors. SK1 activity assays in both cell-free and in vitro settings showed that two compounds were effective SK1 inhibitors. Compound SK-F has potently decreased cancer cell viability in vitro and sensitised mouse breast tumours to docetaxel (DTX) in vivo, without significant whole-body toxicity.ConclusionThrough field template screening, we have identified a new SK1 inhibitor, SK-F, which demonstrated antitumour activity in vitro and in vivo without overt toxicity when combined with DTX.

Correlation between stress drop and applied strain as a biomarker for tumor detection.

This is the first study to measure the viscoelastic behavior of tumor tissues using stepwise compression-relaxation testing, and investigate the measured (Δσ-ε) relation between stress drop (Δσ) and applied strain (ε) as a biomarker for tumor detection. Stepwise compression-relaxation testing was implemented via a 2D tactile sensor to measure stress drop at each applied strain of a sample. Pearson correlation analysis was conducted to quantify the measured Δσ-ε relation as slope of stress drop versus applied strain (m=Δσ/ε) and coefficient of determination (R2). The measured results on soft materials revealed no dependency of coefficient of determination on the testing parameters and dependency of slope on them. Three groups of tissues: five mouse breast tumor (BT) tissues ex vivo, two mouse pancreatic tumor (PT) tissues in vivo and six normal tissues, were measured by using different testing parameters. Coefficient of determination was found to show significant difference among the center, edge and outside sites of all the BT tissues, and no difference between the BT outside sites and the normal tissues. Coefficient of determination also revealed significant difference between before and after treatment of the PT tissues, and no difference between the PT tissues after treatment and the normal tissues. Moreover, coefficient of determination of the PT tissues before treatment was found to be significantly different from that of the BT center sites, but slope failed to capture their difference. Dummy tumors made of silicon rubbers were found to behave differently from the native tumors. By removing the need of fitting the time-dependent data with a viscoelastic model, this study offered a time-efficient solution to quantifying the viscosity for tumor detection.

Abstract LB-297: HER2-targeted antibody drug conjugates induce host immunity and target cancer stem cells and the efficacy can be enhanced by anti-PD-L1

Abstract HER2 represents an ideal target for cancer immunotherapy. HER2-targeted antibody drug conjugate (ADC) was tested in immunocompromised (SCID) host, which doesn't allow the evaluation of the ADC-induced immune responses. In addition, no potential impact of ADC on cancer stem cells (CSCs) has been reported. Furthermore, tumor cells with PD-L1 expression may escape immune attack by converting PD-1 positive immune effector cells into anergy. We hypothesized that HER2-targeted ADC could modulate host immune response, target HER2high CSCs, and the efficacy of ADC could be enhanced by anti-PD-L1. To test this hypothesis, we used two immunocompetent murine models: mouse breast tumor D2/F2 and TNBC 4T1 with enforced expression of HER2, D2F2/E2 and HER2-4T1 respectively. Both D2F2/E2 and HER2-4T1 could generate tumor in immunocompetent Balb/c mice, which allowed us to test the efficacy and specificity of HER2-targeted ADC, and to evaluate host immune responses. In D2F2/E2 mouse model, we found that HER2-targeted ADC significantly inhibited D2F2/E2 tumor growth, but not the parental D2F2 tumors, in a dose dependent manner. The specificity was confirmed in HER2-4T1 model because we observed significant suppression of HER2-targted ADC on HER2-4T1 tumor growth, but not on the parental 4T1 tumor. In parallel, we observed that HER2-targeted ADC treatment reduced the number of HER2 positive cells, and ALDHhigh cells. ALDHhigh cells have been identified as cancer stem cells in multiple malignancies. We have characterized the stemness of ALDHhigh 4T1 cells. In the present study, we confirmed that the ALDHhigh sub-population of D2F2/E2 cells are highly tumorigenic compared to ALDHlow cells. Next, we tested and observed that PD-L1 expression increased in D2F2/E2 and HER2-4T1 fresh tumor cells than in cell lines. Anti-PD-L1 mAb significantly augmented the therapeutic efficacy of HER2-targeted ADC in both D2F2/E2 and HER2-4T1 tumor models. In addition, anti-PD-L1 plus HER2-targeted ADC further reduced the number of HER2 positive cells, and ALDHhigh cells, but increased the number of CD3+ and CD19+ tumor infiltrating lymphocytes (TILs). Expanded CD3+ TILs from the tumor subjected to HER2-targeted ADC plus anti-PD-L1 treatment killed tumor cells significantly more than mono-treatment. Expanded CD19+ TILs from tumor subjected to HER2-targeted ADC plus anti-PD-L1 produced more IgG than all the controls. Importantly, the IgG could specifically bind to tumor cells, resulting in the highest cytotoxicity of the tumor cells via ADCC. Together, our data indicate that HER2-targeted ADC could induce significant host immune responses, which was evidenced by the detection of anti-tumor CD3+ and CD19+ TILs. We first show that HER2-targeted ADC could target ALDHhigh cancer stem cells, and such effect was significantly enhanced by anti-PD-L1 administration. Citation Format: Leiming Xia, Lu Wen, Ming Lin, Comer Frank, Hinrichs Mary Jane, Oberst Michael, Coats Steven, Yangyi Bao, Alfred E. Chang, Max Wicha, Qiao Li. HER2-targeted antibody drug conjugates induce host immunity and target cancer stem cells and the efficacy can be enhanced by anti-PD-L1 [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 LB-297.

Abstract 444: ER Stress marker GRP78 is not expressed on ER-/PR-/Her2- human breast cancer cell surface or secreted

Abstract GRP78 (a Mr 78kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr (i.e., N-linked glycosylation). This important biochemical event is essential for glycoprotein folding and function. When targeted in ER-/PR-/HER2+ breast tumor in athymic nude mice with the N-glycosylation inhibitor tunicamycin, the cancer growth was reduced ~55% in three weeks. Paraffin sections of the tumor exhibited reduced N-glycan level, and reduced angiogenesis but increased GRP78 expression in microvasculature as well as in the tumor tissue (J. Biol. Chem. 286, 29127–29138, 2011). This supported the presence of ER stress. We have now evaluated the effect of tunicamycin on the proliferation of metastatic ER-/PR-/HER2- (MDA-MB-231) and non-metastatic ER+ (MCF-7) human breast cancer cells. Tunicamycin inhibited proliferation of both cells types in a time and dose-dependent manner. Interestingly, GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 µg/ml) treated MDA-MB-231 and in MCF-7 cells and supported by both Western blotting and qPCR. Since GRP78 is an ER stress marker, so we have followed its intracellular localization by immunofluorescence microscopy after treating the cancer cells with tunicamycin as well as after subjecting them to various stress conditions. We have used unfixed cells and stained with either FITC-conjugated Concanavalin A (Con A), or Texas-red conjugated wheat germ agglutinin (WGA) or with anti-GRP antibody. There were expression of N-glycans but not GRP78. GRP78 however became detectable after permeabilization of cells with a brief exposure to ice-cold methanol. GRP78 was not detected in the conditioned media of the cancer cell but a high level of MMP-1 did. We have therefore, concluded that GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. This is obviously, contradicted the current dogma that GRP78 expression on the tumor cell surface interferes with the cancer therapeutic(s) and make them as tumor promoters and not tumor suppressors. Our study, thus, suggests strongly that anti-angiogenic and anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast tumor and all other sold tumors. Supported in part by the university funds and NIH/NIMHD G12MD007583 (KB). Citation Format: Jesus E. Serrano, Andrea Rivera-Ruiz, Krishna Baksi, Dipak K. Banerjee. ER Stress marker GRP78 is not expressed on ER-/PR-/Her2- human breast cancer cell surface or secreted [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 444.

Abstract 3929: The FAD-directed LSD1 specific inhibitor, INCB059872, inhibits cell migration and metastasis by suppressing premetastatic niche formation in a spontaneous metastasis mouse model

Abstract The primary cause of cancer associated mortality is tumor metastasis. The concept of the tumor pre-metastatic niche is supported by evidence of changes at distal pre-metastatic sites that create a permissive environment to allow disseminated tumor cells to seed. Myeloid-derived suppressor cells (MDSCs) remodel the tumor microenvironment and function as immunosuppressive cells to promote tumor growth. Previously, we demonstrated that the clinical stage LSD1 specific inhibitor, INCB059872 significantly reshaped the myeloid compartment in the murine 4T1 syngeneic murine model of breast cancer. Treatment with INCB059872 significantly reduced the population of MDSCs in the tumor microenvironment. Since it has been reported that MDSCs promote establishment of a pre-metastatic niche, we hypothesized that INCB059872 could suppress or delay metastatic processes in the 4T1 model and thereby could impact spontaneous metastases to the lung. In vitro, INCB059872 significantly suppressed cancer cell migration of triple negative breast cancer cells, SUM145PT. In vivo, the effect of INCB059872 on forming the metastatic niche using the 4T1 mouse breast tumor model was explored. Vehicle treated animals exhibited a significant infiltration of MDSCs to the primary tumor and lungs prior to cancer cells metastasizing. In contrast, INCB059872 administration significantly suppressed the infiltration of MDSCs in primary tumor and lung tissues. Histological analyses further demonstrated the reduction of metastatic loci in lung with INCB059872 treatment. Plasma levels of CCL2, a cytokine which is required for the recruitment and functional specialization of MDSCs, were significantly reduced in animals treated with INCB059872. These data suggest a possible mechanism to reduce infiltration of MDSCs into lung tissues. Notably, analyses of molecular pathways using RNA-Seq identified that components of the EMT associated pathway are also downregulated in tumors treated with INCB059872, which further supports the role of INCB059872 in the inhibition of metastasis. Taken together, these preclinical data suggest that inhibition of LSD1 with INCB059872 can suppress metastasis through multiple molecular and cellular mechanisms, notably by inhibition of the formation of the pre-metastatic niche by modulating the population of MDSCs in the primary tumor and distal tissues. Citation Format: Sang Hyun Lee, Melody Diamond, Antony Chadderton, Huiqing Liu, Alla Volgina, Valerie Roman, Michael Weber, Chunhong He, Rebecca Stewart, Denise Hertel, Phillip Liu, Liangxing Wu, Julian Oliver, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. The FAD-directed LSD1 specific inhibitor, INCB059872, inhibits cell migration and metastasis by suppressing premetastatic niche formation in a spontaneous metastasis mouse model [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 3929.

Imaging of IR700DX Labeled Mouse Breast Tumor Using a Custom Angle-Selective Fluorescence Contact Imaging System.

Cancer treatment faces the challenge of identifying small clusters of residual tumor cells in the resection cavity after the gross section of tumor is surgically removed. Despite the introduction of targeted fluorescent probes to guide cancer surgeries, large, bulky, optical components restrict the ability of fluorescence imaging devices to detect small clusters of tumor cells in the complex surgical cavity. We have developed a small size-scale contact fluorescence image sensor that incorporates angle-selective gratings and a thin 15 m amorphous silicon optical wavelength filter for detecting residual cancer tissue in vivo. Using a custom fluorescent probe combining a fluorescent dye, IR700DX, with a targeted antibody, Trastuzumab, we label and visualize breast tissue in in vivo mouse models of breast cancer. When imaging tumorbearing mice injected with the probe, HER2+ breast cancer tissue intensity is 3.80.8 times brighter than other tissue. Excised cancer tumors and residual cancer attached to healthy tissue are imaged using the custom image sensor. Residual cancer tissue can be detected in real-time and is imaged with a high SNR of 45 dB using an integration time of only 40 ms.

Tumours trigger systemic disruption.

Breast tumours in mice promote metabolic changes in the liver and sleep disruption through increasing activity of hypocretin (also known as orexin) neurons (HO neurons).

Plasmonic Optical Imaging of Gold Nanorods Localization in Small Animals

Gold nanoparticles (GNP) have been intensively investigated for applications in cancer imaging and therapy. Most imaging studies focused on microscopic imaging. Their potential as optical imaging probes for whole body small animal imaging has rarely been explored. Taking advantage of their surface plasmon resonance (SPR) properties, we aim to develop a noninvasive diffuse optical imaging method to map the distribution of a special type of GNP, gold nanorods (GNR), in small animals. We developed an integrated dual-modality imaging system capable of both x-ray computed tomography (XCT) and diffuse optical tomography (DOT). XCT provides the animal anatomy and contour required for DOT; DOT maps the distribution of GNR in the animal. This SPR enhanced optical imaging (SPROI) technique was investigated using simulation, phantom and mouse experiments. The distribution of GNR at various concentrations (0.1–100 nM, or 3.5 ug/g–3.5 mg/g) was successfully reconstructed from centimeter-scaled volumes. SPROI detected GNR at 18 μg/g concentration in the mouse breast tumor, and is 3 orders more sensitive than x-ray imaging. This study demonstrated the high sensitivity of SPROI in mapping GNR distributions in small animals. It does not require additional imaging tags other than GNR themselves. SPROI can be used to detect tumors targeted by GNR via passive targeting based on enhanced permeability and retention or via active targeting using biologically conjugated ligands.

Application of polymersomes engineered to target p32 protein for detection of small breast tumors in mice.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as ∼20 mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.

Metabolomics of oncogene-specific metabolic reprogramming during breast cancer

BackgroundThe complex yet interrelated connections between cancer metabolism and oncogenic driver genes are relatively unexplored but have the potential to identify novel biomarkers and drug targets with prognostic and therapeutic value. The goal of this study was to identify global metabolic profiles of breast tumors isolated from multiple transgenic mouse models and to identify unique metabolic signatures driven by these oncogenes.MethodsUsing mass spectrometry (GC-MS, LC-MS/MS, and capillary zone electrophoresis (CZE)-MS platforms), we quantified and compared the levels of 374 metabolites in breast tissue from normal and transgenic mouse breast cancer models overexpressing a panel of oncogenes (PyMT, PyMT-DB, Wnt1, Neu, and C3-TAg). We also compared the mouse metabolomics data to published human metabolomics data already linked to clinical data.ResultsThrough analysis of our metabolomics data, we identified metabolic differences between normal and tumor breast tissues as well as metabolic differences unique to each initiating oncogene. We also quantified the metabolic profiles of the mammary fat pad versus mammary epithelium by CZE-MS/MS. However, the differences between the tissues did not account for the majority of the metabolic differences between the normal mammary gland and breast tumor tissues. Therefore, the differences between the cohorts were unlikely due to cellular heterogeneity. Of the mouse models used in this study, C3-TAg was the only cohort with a tumor metabolic signature composed of ten metabolites that had significant prognostic value in breast cancer patients. Gene expression analysis identified candidate genes that may contribute to the metabolic reprogramming.ConclusionsThis study identifies oncogene-induced metabolic reprogramming within mouse breast tumors and compares the results to that of human breast tumors, providing a unique look at the relationship between and clinical value of oncogene initiation and metabolism during breast cancer.

Abstract P1-08-02: Breast tumor-specific MHC-II expression drives a unique pattern of adaptive resistance to antitumor immunity through MHC-II receptor checkpoint engagement

Abstract Background: We have previously shown that some breast cancers express major histocompatibility complex II (MHC-II), correlating with enhanced immune infiltration. In other tumor types, we have shown that MHC-II expression on tumor cells predicts clinical response to checkpoint inhibition. We sought to determine the direct effects of MHC-II on anti-tumor immunity and characterize mechanisms of immune escape in this breast cancer subset. Methods: To determine the functional effects of MHC-II on tumor cells, we generated isogenic mouse breast tumor cells with enforced MHC-II expression and determined their ability to generate tumors in syngeneic mice, the impact on immunity, and their response to checkpoint inhibition. In a series of molecularly-characterized HER2+ (n=8) and triple-negative breast cancers (TNBC; n=103), we performed immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) for Lag-3, PD-L1, CD4, CD8, FCRL6, and granzyme B. Results: Following injection in syngeneic immunocompetent mice, MHC-II+ mouse breast tumors were more frequently rejected (p=0.04) and recruited greater numbers of CD4+ TILs. When MHC-II+ tumors escaped rejection, they expressed higher degrees of PD-1 and Lag-3 in the tumor and in the draining lymph node. Since Lag-3 is a checkpoint that specifically targets MHC-II, we hypothesized that MHC-II+ breast cancers escape anti-tumor immunity through suppressing MHC-II-mediated antigen presentation. Combinations of anti-Lag-3 and anti-Pd-1 antibodies inhibited growth of MHC-II+ tumors. These findings led us to also explore Fc receptor-like 6 (FCRL6), a previously reported MHC-II receptor expressed on NK and cytotoxic T cells. Residual MHC-II+ TNBC post-neoadjuvant chemotherapy (NAC) recruited greater numbers of CD4+ and CD8+ TILs (p=0.0001 and p=0.0002), suggesting enhanced immune recognition. However, MHC-II+ TNBCs also demonstrated a greater frequency of Lag-3+ and FCRL6+ TILs (p<0.001 and p=0.01, respectively) which frequently co-occurred (p=0.003). Thus, our data suggest that MHC-II expression in breast tumors supports recruitment of MHC-II-specific checkpoint-positive TILs. In line with this concept, QIF analysis demonstrated that the presence of Lag3+ and/or FCRL6+ TILs was strongly associated with suppression of T cell cytotoxicity as assessed by granzyme-B+ CD8+ T cells (p=0.0001 and p=0.002, respectively). Functional analyses of FCRL6 on human NK cell lines and peripheral blood mononuclear cells (PBMCs) demonstrated that like Lag3, FCRL6 is a checkpoint which engages MHC-II and suppresses cytotoxic NK and T cell activity. Conclusions: These data suggest that MHC-II+ breast tumors are immunologically active and circumvent anti-tumor immunity by targeting MHC-II antigen presentation through recruitment of Lag-3+ and FCRL6+ TILs. We describe herein FCRL6 as a novel bona fide immune checkpoint which targets MHC-II, which may impact a variety of cancers. MHC-II expression status may be a useful biomarker for patient stratification on anti-PD-1/anti-Lag-3 combination, and eventually, anti-PD-1/anti-FCRL6 combinations in patients with breast cancer. Citation Format: Balko JM, Johnson DB, Ericsson-Gonzalez P, Nixon MJ, Salgado R, Sanchez V, Shreeder DM, Rimm DL, Loi S, Kim JY, Bordeaux J, Sanders ME, Davis RS. Breast tumor-specific MHC-II expression drives a unique pattern of adaptive resistance to antitumor immunity through MHC-II receptor checkpoint engagement [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-08-02.

Abstract GS1-05: Tandem duplicator phenotypes define 50% of triple negative breast cancers

Abstract Background. We recently discovered a unique chromotype, the Tandem Duplicator Phenotype (TDP), characterized by hundreds of somatic tandem duplications (TDs) scattered throughout the genome of a large percentage of triple negative breast cancers (TNBCs). Importantly, we observed that the TDP associates with a better response to cisplatin therapy in vitro and in vivo, suggesting that it is a tractable and quantitative biomarker of response to platinum-based therapy. Here, we expand on our initial findings by analyzing Whole-Genome (WG) sequences of over 2,700 tumors. Methods. TD coordinates from WG sequences relative to 2717 tumors were assembled from over 30 independent studies representing several cancer types, including 254 TNBCs. WG sequencing of mouse breast tumors was carried out using standard Illumina protocols. The number, distribution and span-size of somatic TDs from a training set of 992 tumors were used to develop a TDP classifier that identifies highly recurrent but clearly distinct TDP profiles. The TDP classifier was then applied to the remaining tumor sequences. WG mutation and copy number datasets were investigated to identify the genetic drivers associated with each TDP profile, and the genomic consequences of different TDPs were evaluated through identification of genomic hotspots for gene duplication and transection. Results. We describe six different TDPs featuring distinct TD span size distributions, with peaks at 10Kb (group 1), 300Kb (group 2) and 3Mb (group 3), or different combinations of these (mix12, mix13 and mix23). More than half of all TNBC display a TDP. Of these, 55% classify as group 1, 14% as group 2 and 15% as group mix12. Whereas all TDP groups show a higher TP53 mutation rate compared to non-TDP tumors, each TDP profile is characterized by specific additional gene perturbations, with loss of BRCA1 occurring in groups 1, mix12 and mix13; CCNE1 amplification in group 2; and CDK12 mutations in group mix23. We show that different TDPs are subject to the perturbation of specific oncogenic networks resulting from the duplication of oncogenes by larger TDs (>300Kb) or the disruption of tumor suppressors via double transections by shorter TDs (10Kb). Indeed, tumor suppressor genes such as PTEN, RB1 and MLL3 are frequently disrupted by TDs in TNBC TDP group 1 tumors, whereas TNBC TDP group 2 tumors commonly feature duplication of oncogenes such as MYC and MALAT1. Finally, through WG analyses of 18 mouse models (GEMMs) of breast cancer, we provide the first mechanistic evidence of the driving role of conjoint loss of TP53 and BRCA1, but not of BRCA2, in inducing the TDP group 1 profile. Conclusions. Our study shows a definitive genetic induction of one specific form of TDP (group 1) characterized by 10kb TD span. Different TDP profiles are characterized by alternative somatic genetic origins but always couple with disruptive TP53 mutations. The consequences of the massive TD formation in TDP TNBCs suggest a systems strategy to tumor induction involving heterogeneous combinations of oncogenes and tumor suppressors. That these TDP forms, accounting for ˜50% of TNBC, are associated with significant sensitivity to cisplatin suggest that this chromotype may identify TNBC patients who would benefit from upfront platinum-based chemotherapy. Citation Format: Liu ET, Menghi F, Barthel F, Yadav V, Tang M, Ji B, Carter G, Jonkers J, Verhaak R. Tandem duplicator phenotypes define 50% of triple negative breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-05.

Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media.

GRP78 (an Mr 78 kDa calcium dependent glucose binding protein) is located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER-/PR-/HER2+ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER-/PR-/HER2-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Nonmetastatic estrogen receptor positive (ER+) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/mL) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A) or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

Taking aim at regulatory T cells

Cancer Immunotherapy Cancer immunotherapy attempts to stimulate the patient's immune system against a tumor, but it can be limited by the suppressive effects of the patient's own regulatory T (Treg) cells. Nie et al. showed that coinhibiting a TNF (tumor necrosis factor) receptor reduced Treg cell activity and proliferation, stimulated antitumor immune memory, and even shrank colon and breast tumors in mice that were unresponsive to common single-agent immunotherapies. Thus, adding anti-TNF therapeutics may help to increase and broaden the efficacy of immunotherapy for cancer patients. Sci. Signal. 10 , eaan0790 (2018).

Bio-transformation of Graphene Oxide in Lung Fluids Significantly Enhances Its Photothermal Efficacy.

Rationale: Graphene oxide (GO) has shown great promises in biomedical applications, such as drug delivery and thermotherapeutics, owing to its extraordinary physicochemical properties. Nonetheless, current biomedical applications of GO materials are premised on the basis of predesigned functions, and little consideration has been given to the influence of bio-transformation in the physiological environment on the physicochemical properties and predesigned functionalities of these materials. Hence, it is crucial to uncover the possible influence on GO's physicochemical properties and predesigned functionalities for better applications.Methods: Bio-transformed GOs were characterized by X-ray diffraction (XRD) spectra, Raman spectra, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared transmission (FT-IR) spectra. The morphologies of various GO materials were assessed via transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) images. The photothermal (PTT) performance of different GO materials in vitro and in vivo were measured using 808 nm laser at a power density of 2 W/cm2. The PTT efficacy was determined using transplanted 4T1 cells-derived breast tumors in mice.Results: Bio-transformation of GO in the lung (a main target organ for GO to localize in vivo) can induce dramatic changes to its physicochemical properties and morphology, and consequently, its performances in biomedical applications. Specifically, GO underwent significant reduction in two simulated lung fluids, Gamble's solution and artificial lysosomal fluid (ALF), as evidenced by the increase of C/O ratio (the ratio of C content to O content) relative to pristine GO. Bio-transformation also altered GO's morphology, characterized by sheet folding and wrinkle formation. Intriguingly, bio-transformation elevated the PTT performance of GO in vitro, and this elevation further facilitated PTT-based tumor-killing efficacy in tumor cells in vitro and in a mouse model with transplanted tumors. Bio-transformation also compromised the interaction between drug with GO, leading to reduced drug adsorption, as tested using doxorubicin (DOX).Conclusions: Transformation in Gamble's solution and ALF resulted in varied degrees of improved performances of GO, due to the differential effects on GO's physicochemical properties. Our findings unveiled an overlooked impact of GO bio-transformation, and unearthed a favorable trait of GO materials in thermotherapeutics and drug delivery in the lung microenvironment.

2D Superparamagnetic Tantalum Carbide Composite MXenes for Efficient Breast-Cancer Theranostics.

Background: The emergence of two-dimensional MXenes has spurred their versatile applications in broad fields, but the exploring of novel MXene-based family members and their potential applications in theranostic nanomedicine (concurrent diagnostic imaging and therapy) have been rarely explored. In this work, we report the construction of a novel superparamagnetic MXene-based theranostic nanoplatform for efficient breast-cancer theranostics, which was based on intriguing tantalum carbide (Ta4C3) MXene and its further rational surface-superparamagnetic iron-oxide functionalization (Ta4C3-IONP-SPs composite MXenes) for efficient breast-cancer theranostic.Methods: The fabrication of ultrathin Ta4C3 nanosheets was based on an exfoliation strategy and superparamagnetic iron oxide nanoparticles were in-situ grown onto the surface of Ta4C3 MXene according to the redox reaction of MXene. Ta4C3-IONP MXenes were modified with soybean phospholipid (SP) to guarantee high stability in physiological conditions. The photothermal therapy, contrast-enhanced CT, T2-weighted magnetic resonance imaging and the high biocompatibility of these composite nanosheets have also been evaluated in vitro at cellular level and in vivo on mice breast tumor allograft tumor model.Results: The Ta component of Ta4C3-IONP-SPs exhibits high performance for contrast-enhanced CT imaging because of its high atomic number and high X-ray attenuation coefficient, and the integrated superparamagnetic IONPs act as excellent contrast agents for T2-weighted magnetic resonance imaging. Especially, these Ta4C3-IONP-SPs composite nanosheets with high photothermal-conversion efficiency (η: 32.5%) has achieved complete tumor eradication without reoccurrence, verifying their highly efficient breast-tumor photo-ablation performance.Conclusion: This work not only significantly broadens the biomedical applications of MXene-based nanoplatforms (Ta4C3 MXene) by exploring their novel family members and further functionalization strategies (magnetic functionalization in this work), but also provides a novel and efficient theranostic nanoplatform for efficient breast-cancer theranostics.

JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

Cancer stem cells (CSCs) are critical for cancer progression and chemoresistance. How lipid metabolism regulates CSCs and chemoresistance remains elusive. Here, we demonstrate that JAK/STAT3 regulates lipid metabolism, which promotes breast CSCs (BCSCs) and cancer chemoresistance. Inhibiting JAK/STAT3 blocks BCSC self-renewal and expression of diverse lipid metabolic genes, including carnitine palmitoyltransferase 1B (CPT1B), which encodes the critical enzyme for fatty acid β-oxidation (FAO). Moreover, mammary-adipocyte-derived leptin upregulates STAT3-induced CPT1B expression and FAO activity in BCSCs. Human breast-cancer-derived data suggest that the STAT3-CPT1B-FAO pathway promotes cancer cell stemness and chemoresistance. Blocking FAO and/or leptin re-sensitizes them to chemotherapy and inhibits BCSCs in mouse breast tumors invivo. We identify a critical pathway for BCSC maintenance and breast cancer chemoresistance.

Proteomic Profiling of Exosomes Secreted by Breast Cancer Cells with Varying Metastatic Potential.

Cancer cells actively release extracellular vesicles, including exosomes, into the surrounding microenvironment. Exosomes play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteome profile of exosomes isolated from cells with different metastatic potential and the role of these exosomes in driving metastasis remains unclear. Here, we conduct a comparative proteomic analysis of exosomes isolated from several genetically related mouse breast tumor lines with different metastatic propensity. The amount of exosomes produced and the extent of cancer-associated protein cargo vary significantly between nonmetastatic and metastatic cell-derived exosomes. Metastatic cell-derived exosomes contain proteins that promote migration, proliferation, invasion, and angiogenesis while the nonmetastatic cell-derived exosomes contain proteins involved in cell-cell/cell-matrix adhesion and polarity maintenance. The metastatic exosomes contain a distinct set of membrane proteins including Ceruloplasmin and Metadherin which could presumably aid in targeting the primary cancer cells to specific metastatic sites. Hence, it can be concluded that the exosomes contain different protein cargo based on the host cells metastatic properties and can facilitate in the dissemination of the primary tumors to distant sites.