SUM149 Inflammatory Breast Cancer Research Articles

Abstract PO2-24-03: Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer known for its rapid progression and high metastatic potential without known distinctive drivers. Currently, there are no FDA-approved targeted therapies specifically for IBC patients, highlighting the urgent need for novel and effective treatments. Through our investigation of metastatic xenograft IBC sublines, we have identified soluble E-cadherin (sEcad), an extracellular proteolytic fragment of full-length E-cadherin, as a protein associated with IBC tumor progression. We hypothesize that sEcad promotes IBC tumorigenesis by suppressing ferroptosis. Methods: MDA-IBC3 (ER–/HER2+) and SUM149 (ER–/HER2–) IBC cell lines were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Cell death for ferroptosis was measured by propidium iodide staining using flow cytometry. Mass spectrometry and Bio-ID-based proteomics assays were used to identify sEcad-interacting proteins and potential mechanisms. For in vivo studies, control and sEcad -overexpressing SUM149 and MDA-IBC3 cells were injected into the cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Serum sEcad levels from IBC patients (n=301) were analyzed by ELISA. Results: High serum sEcad levels in IBC patients correlated with poorer OS (p=0.02) and earlier development of metastasis (p=0.006). Overexpression of sEcad in IBC cell lines enhanced cell proliferation and colony formation in vitro. Mice injected with sEcad-overexpressing SUM149 and MDA-IBC3 cells had significantly higher tumor growth rates compared to controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, mass spectrometry and Bio-ID assays identified Protein Disulfide Isomerase Family A Member 4 (PDIA4) as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Additionally, sEcad increased the expression of PDIA4, an enzyme that plays a role in cellular redox regulation and protein folding. PDIA4 has been shown to regulate key players of ferroptosis such as ATF4, SLC7A11. Our results showed that, compared to controls, sEcad-overexpressing IBC cells displayed significant resistance to ferroptosis inducers, including RSL3, FIN56, and sulfasalazine. Knockdown of PDIA4 in sEcad high-expressing cells significantly increased their sensitivity to ferroptosis-promoting drugs. Moreover, we found that PDIA4 regulates ATF4 to impact ferroptosis-mediated cell death in IBC cells. Conclusions: Our study shows that sEcad affects ferroptosis through PDIA4, impacting tumorigenesis in IBC. These findings uncover a novel and pivotal role for sEcad in IBC tumor growth and progression, providing new insights and potential therapeutic targets for IBC patients. Citation Format: Xiaoding Hu, Yun Xiong, Emilly Villodre, Juhee Song, Debu Tripathy, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-03.

XIAP overexpressing inflammatory breast cancer patients have high infiltration of immunosuppressive subsets and increased TNFR1 signaling targetable with Birinapant

ObjectiveTo assess the expression pattern of X-linked inhibitor of apoptosis protein (XIAP), a cellular stress sensor, and delineate the associated changes in the tumor immune microenvironment (TiME) for prognostic value and new therapeutic targets in inflammatory breast cancer (IBC). MethodsImmunohistochemistry was conducted to assess the spatial localization of immune subsets, XIAP, and PDL1 expression in IBC and non-inflammatory breast cancer (nIBC) pretreatment tumors (n = 142). Validation and further exploration were performed by gene expression analysis of patient tumors along with signaling studies in a co-culture model. ResultsHigh XIAP in 37/81 IBC patients correlated significantly with high PD-L1, increased infiltration of FOXP3+ Tregs, CD163+ tumor-associated macrophages (TAMs), low CD8/CD163 ratio in both tumor stroma (TS) and invasive margins (IM), and higher CD8+ T cells and CD79α+ B cells in the IM. Gene set enrichment analysis identified cellular stress response- and inflammation-related genes along with tumor necrosis factor receptor 1 (TNFR1) expression in high-XIAP IBC tumors. Induction of TNFR1 and XIAP was observed when patient-derived SUM149 IBC cells were co-cultured with human macrophage-conditioned media simulating TAMs, further demonstrating that the TNF-α signaling pathway is a likely candidate governing TAM-induced XIAP overexpression in IBC cells. Finally, addition of Birinapant, a pan IAP antagonist, induced cell death in the pro-survival cytokine-enriched conditions. ConclusionUsing immunophenotyping and gene expression analysis in patient biospecimens along with in silico modeling and a preclinical model with a pan-IAP antagonist, this study revealed an interplay between increased TAMs, TNF-α signaling, and XIAP activation during (immune) stress in IBC. These data demonstrate the potential of IAP antagonists as immunomodulators for improving IBC therapeutic regimens.

High-fat diet, but not duration of lactation, increases mammary gland lymphatic vessel function and subsequent growth of inflammatory breast cancer cells

Inflammatory breast cancer (IBC) presents as rapid-onset swelling and breast skin changes caused by tumor emboli in the breast and breast skin lymphatics. IBC has been linked with obesity and duration of breastfeeding, but how these factors affect IBC tumor progression is not clear. We modeled the simultaneous effects of diet and weaning in mice on in vivo lymphatic function; on IBC tumor growth; and on aspects of the mammary gland microenvironment before and after IBC (SUM149) xenograft inoculation. We hypothesized that weaning status and diet would have synergistic effects on lymphatic function and the breast microenvironment to enhance IBC tumor growth. Changes in lymphatic structure and function were characterized with in vivo near-infrared fluorescence (NIRF) imaging. Mice were fed either a high-fat diet (HFD; 60 kcal%) or a normal/low-fat diet (LFD; 10 kcal%), bred twice, and subjected to either normal-duration nursing (NW) or forced weaning (FW). SUM149 IBC tumors were implanted at 14 months; images were obtained before and after implantation. Multiparous mice fed HFD showed increased pre-tumor lymphatic pulsing in both the FW and NW groups relative to mice fed LFD. HFD promoted tumor growth independent of weaning time (P = 0.04). Pre-tumor lymphatic pulsing was associated with tumor volume at 8 weeks (P = 0.02) and was significantly correlated with expression of the lymphatic tracking ligand CCL21 (P = 0.05, Table 1). HFD significantly increased the numbers of monocyte-derived IBA1+, CD163+, and CD11c+ cells (P < 0.0001, P < 0.0001, P = 0.0005) in the contralateral, non-tumor-bearing mammary gland. Numbers of lymphangiogenic podoplanin+/IBA1+ macrophages were increased in the ducts of HFD and FW mice (all P < 0.003). HFD in nulliparous mice had a similar increase in lymphatic pulsing at 14 weeks (P = 0.006), indicating that this functional change was independent of parity. We conclude that HFD induced increases in mammary gland lymphatic function, assessed as pulsing rate before tumor initiation, and correlated with inflammation in the mammary gland and increased SUM149 tumor growth. The relationship between diet, lymphatic pulsing, and tumor growth warrants further investigation.

Metadherin Regulates Inflammatory Breast Cancer Invasion and Metastasis.

Inflammatory breast cancer (IBC) is one of the most lethal subtypes of breast cancer (BC), accounting for approximately 1-5% of all cases of BC. Challenges in IBC include accurate and early diagnosis and the development of effective targeted therapies. Our previous studies identified the overexpression of metadherin (MTDH) in the plasma membrane of IBC cells, further confirmed in patient tissues. MTDH has been found to play a role in signaling pathways related to cancer. However, its mechanism of action in the progression of IBC remains unknown. To evaluate the function of MTDH, SUM-149 and SUM-190 IBC cells were edited with CRISPR/Cas9 vectors for in vitro characterization studies and used in mouse IBC xenografts. Our results demonstrate that the absence of MTDH significantly reduces IBC cell migration, proliferation, tumor spheroid formation, and the expression of NF-κB and STAT3 signaling molecules, which are crucial oncogenic pathways in IBC. Furthermore, IBC xenografts showed significant differences in tumor growth patterns, and lung tissue revealed epithelial-like cells in 43% of wild-type (WT) compared to 29% of CRISPR xenografts. Our study emphasizes the role of MTDH as a potential therapeutic target for the progression of IBC.

Abstract PD10-08: Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no targeted therapy has been approved specifically for this disease. There is a critical need for innovative treatment approaches for patients with IBC. There is a paucity of data on immune checkpoint inhibitors (ICIs) in IBC, yet a strong biological rationale exists to lay the groundwork for testing the efficacy of ICIs in IBC. We have previously shown that an anti–epidermal growth factor receptor (EGFR) antibody, panitumumab (sponsored by Amgen), combined with Abraxane (sponsored by Bristol Myers Squibb), and carboplatin followed by 5-fluorouracil, epirubicin, and cyclophosphamide achieves a very high pathologic complete response (42%) in patients with triple-negative receptor status (TN-IBC) (NCT01036087). We hypothesize that inducing a broad shift in the IBC tumor microenvironment (TME), a critical driver of the IBC clinical phenotype and metastasis, from an immunosuppressive to an immunoreactive phenotype can enhance the efficacy of ICIs. Here we report the impact of targeting EGFR on ICI effectiveness by modulating the immunosuppressive TME in TN-IBC. Methods: We examined the effects of panitumumab on components of the immune TME in IBC patient tissues collected before and after panitumumab treatment. Furthermore, we analyzed panitumumab effects in an IBC SUM149 humanized mouse model (SUM149-hu-NSG-SGM3) by using multiplex immunofluorescence staining (mIF) of single-cell RNA-sequencing (scRNA-seq) and multicolor flow cytometry. We examined the changes in chemokines in humanized mouse tissue after panitumumab treatment using a cytokine antibody array and enzyme-linked immunosorbent assay (ELISA). We also studied the mechanism of EGFR regulation of immunosuppressive chemokine expression via transcription factor EGR1 by using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and chromatin immunoprecipitation (ChIP) assays. In addition, we tested the efficacy of panitumumab combined with anti-PD-L1 antibody (Bio X Cell, clone 29E.2A3) in reducing IBC tumor growth in a humanized IBC SUM149 mouse model. Results: IBC patients who achieved pathologic complete response after receiving panitumumab/neoadjuvant chemotherapy had increased levels of CD8+ T-cells and decreased levels of M2 macrophages and T-reg cells. In the humanized mouse model, panitumumab treatment reduced IBC tumor growth and increased immune response that favors an antitumor effect, as shown by scRNA-seq, flow cytometry, and mIF analyses. In addition, we observed that panitumumab treatment increased the expression of chemokines that function as a chemoattractant for CD8+ T-cells, including CXCL10, CCL4, and CXCL9. We also observed that panitumumab treatment reduced EGR1 expression, leading to the downregulation of chemokines involved in the recruitment of immunosuppressive M2 macrophages and T-reg cells, including CCL2, CCL20 CXCL5, and IL-8. Finally, panitumumab enhanced the reduction in tumor growth by anti-PD-L1 antibody treatment; furthermore, the combination of panitumumab and anti-PD-L1 antibody increased the presence of CD8+ T-cells and reduced the presence of T-reg cells and M2 macrophages more effectively than either treatment alone. Conclusion: Panitumumab treatment converts the immunosuppressive IBC TME to an immunoreactive status by modulating the global expression of chemokines by downregulating the EGR1 transcription factor. This modulation of the TME by panitumumab improves the inhibition of IBC tumor growth by an ICI. Our study is the first to demonstrate that targeting the EGFR/EGR1 axis remodels the IBC TME by regulating chemokine expression, thus boosting the antitumor immune response in IBC and response to ICI. Citation Format: Xiaoping Wang, Takashi Semba, Ganiraju C. Manyam, Jing Wang, Shan Shao, Francois Bertucci, Pascal Finetti, Savitri Krishnamurthy, Lan ThiHanh Phi, Troy Pearson, Jared K. Burks, Evan N. Cohen, James M. Reuben, Fei Yang, Hu Min, Nicholas Navin, Toshiaki Iwase, Yichao Shen, Xiang Zhang, Debu Tripathy, Naoto T. Ueno. Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-08.

Abstract P1-24-06: Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression

Abstract Background. Lymphovascular invasion (LVI) and breast tumor emboli within dermal and breast lymphatic vessels are prognostic for metastatic spread and poor outcomes, and are abundant in Inflammatory breast cancer (IBC). IBC is an aggressive breast cancer that presents suddenly with breast swelling and redness due to tumor emboli in lymphatics. Lack of breast-feeding and obesity are IBC risk factors. We sought to demonstrate the combinatorial effects of a high-fat diet and nursing on lymphatic function and compare these to IBC tumor induced changes in lymphatic function. We hypothesize that risk factors for aggressive breast cancer may alter lymphatic function in the normal gland prior to tumor initiation. Methods. Following two rounds of pregnancy in 20 multiparous SCID Beige immunocompromised mice, half of the mice were force weaned while half nursed pups. Prior to forced weaning, half of each of these groups were fed a high fat diet (HFD: 60 Kcal %, N = 10) while the other half received a low-fat diet (LFD: 10 Kcal %, N = 10). Consecutive dynamic near-infrared fluorescence (NIRF) lymphatic imaging was performed at 6-7 months (covid interruption) and 14 months after initiating the diet by injecting a near-IR fluorophore into the mammary fatpad and recording lymphatic pulsing over 8 minutes using V++. Matlab and ImageJ were used to quantify pulsing rates on the ventral lymphatics in each animal. Fatpads were subsequently inoculated with SUM149 IBC cells and imaging was repeated 16 months post diet initiation. Lymphatic imaging over time by HFD vs LFD was further studied in nulliparous animals. Tissues were collected for further analyses. ResultsData analysis prior to tumor injection, demonstrated lymphatic pulsing (pulses/4 minutes) increased over time in HFD force weaned (HFFW) and HFD nursing (HFN) animals only (65.5 vs 72.6, P=0.059; 60.1 vs 76.6, P=0.0099, respectively). Comparing HFFW and HFN to matched LFD groups (LFFW and LFN), at 14 weeks HFD was associated with increased pumping after forced weaning (62.3 vs. 72.6, P=0.074), and nursing (62.5 vs 76.6, P=0.0023). There was an increase in pulsing after tumor initiation (16 months after initiation of diet) in all groups (80.1, 84.1, 83.2, 82.4, P &amp;gt; 0.05 all comparisons to initial timepoint). In a separate experiment examining HFD (N=5) vs LFD (N=5) in nulliparous mice, lymphatic contractile activity increased in all animals over. time, average ventral lymphatic contractile frequency for LFD and HFD at week 8,11 and 14 weeks after diet initiation were 5, 8.64, 15.9 pumps/4 mins vs 11.8, 18.5, 28.2 pumps/4 mins, (P = 0.01, 0.05, and 0.0005 respectively). ConclusionsHFD increased lymphatic pulsing rate over time to a significantly greater extent than LFD continuing over 14 months independent of reproductive and nursing status. Tumor initiation prompted further increased pulsing rates beyond that observed after HFD across all groups. The magnitude of the effect of HFD on lymphatic pulsing approached the rate after tumor initiation, while reproductive variables did not impact lymphatic pulsing. Further studies are warranted to demonstrate the relationship if any between lymphatic pumping pre-initiation and LVI after tumor initiation and examine the role of intervention on reducing LVI. Citation Format: Wintana Balema, Janelle Morton, Richard Larson, Fred C Velasquez, Eva Sevick-Muraca, Wendy A Woodward. Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-06.

Abstract PS19-07: Plasma exosomal miRNAs: A minimally invasive diagnostic biomarker for inflammatory breast carcinoma

Abstract Background. Inflammatory Breast Cancer (IBC) is a rare, but aggressive subtype of breast carcinoma with dermal lymphatic invasion in young females. IBC is poorly diagnosed as it develops vast rapidly relative to other non-IBC and without distinct lumps formation, moreover, it is usually misdiagnosed with mastitis. Nanosized exosomes (20-200nm) circulating in liquid biopsies are a promising minimally invasive diagnostic alternative to standard biopsies. In cancer microenvironment, they modulate cancer progression via shuttling their encapsulated microRNAs (miRNAs) into recipient cells to either trigger signaling or induce malignant transformation of targeted cells. While small non-coding regulatory miRNAs are an already known cancer biomarkers, exosomal miRNAs serve as a novel class of diagnostic biomarkers. The present study aims to evaluate the expression levels of exosomal miRNAs relevant to IBC pathogenesis. Design/Materials/Methods. This is a prospective case-control study that includes 77 females; 57 were diagnosed with breast cancer (34 non-IBC and 23 IBC), while 20 were healthy volunteers. All have signed an informed consent to be enrolled in this investigation. Patients included were neither pregnant nor issued with bloodborne or autoimmune disease, while healthy subjects had no oncologic history. Plasma circulating exosomes were isolated using precipitation and ultracentrifugation methods. The successful isolation was verified by dynamic light scattering (DLS), transmission electron microscopy (TEM), Western blot (WB), and Dot blot (DB). The expression level of exosomes-derived miR-181b-5p, miR-222-5p and let-7a was quantified by qPCR. Afterwards, that expression level was verified in the human non-IBC MDA-MB-231 and IBC SUM149 cell lines. Statistics. Difference between groups was tested using Student’s t-test through IBM SPSS statistics software package, version 24. A p &amp;lt; 0.05 was considered significant. Results. DLS and TEM analysis revealed that nanovesicles with 146.65 nm were successfully isolated. Moreover, CD63, HSP70, Alix90 and GM130 antibodies used in DB and WB have further confirmed the successful isolation. Relative to non-IBC, our qPCR showed that plasma exosomes-derived miR-181b-5p and miR-222-5p were significantly (p &amp;lt;0.0001 and p &amp;lt;0.01, respectively) upregulated in IBC, whereas exosomal let-7a was significantly (p &amp;lt;0.0001) downregulated in IBC. Further, the expression levels of miR181-5p and miR-222 were upregulated in SUM-149-derived exosomes relative to those of MDA-MB-231-derived exosomes ( Both p &amp;lt; 0.001), while the expression levels of let-7a did not significantly change. Diagnostic accuracy of the candidate plasma exosomal miRNAs was assessed via receiver operating characteristic (ROC) curves analysis. Interestingly, Area Under Curve (AUC) was 0.826 for miR-181-5p (p &amp;lt;0.0001), 0.7212 for miR-222-5p (p &amp;lt;0.0001), and 0.9188 for let7a (p &amp;lt;0.01). Additionally, bioinformatical analysis for predicted targets of the identified exosomal miRNAs will be assessed. Conclusion. Exosomes derived miR-181-5p, miR-222-5p and let7-7a may act as non-invasive diagnostic biomarkers to discriminate IBC from non-IBC patients. Citation Format: Sarah Hamdy Ahmed, Ahmed El-Damen, Mohamed A. Badawy, Mohamed El-Shinawi, Martin Götte, Sherif Abdelaziz Ibrahim. Plasma exosomal miRNAs: A minimally invasive diagnostic biomarker for inflammatory breast carcinoma [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 PS19-07.

Infrared Microspectroscopy and Imaging Analysis of Inflammatory and Non-Inflammatory Breast Cancer Cells and Their GAG Secretome.

Glycosaminoglycans (GAGs)/proteoglycans (PGs) play a pivotal role in the metastasis of inflammatory breast cancer (IBC). They represent biomarkers and targets in diagnosis and treatment of different cancers including breast cancer. Thus, GAGs/PGs could represent potential prognostic/diagnostic biomarkers for IBC. In the present study, non-IBC MDA-MB-231, MCF7, SKBR3 cells and IBC SUM149 cells, as well as their GAG secretome were analyzed. The latter was measured in toto as dried drops with high-throughput (HT) Fourier Transform InfraRed (FTIR) spectroscopy and imaging. FTIR imaging was also employed to investigate single whole breast cancer cells while synchrotron-FTIR microspectroscopy was used to specifically target their cytoplasms. Data were analyzed by hierarchical cluster analysis and principal components analysis. Results obtained from HT-FTIR analysis of GAG drops showed that the inter-group variability enabled us to delineate between cell types in the GAG absorption range 1350–800 cm−1. Similar results were obtained for FTIR imaging of GAG extracts and fixed single whole cells. Synchrotron-FTIR data from cytoplasms allowed discrimination between non-IBC and IBC. Thus, by using GAG specific region, not only different breast cancer cell lines could be differentiated, but also non-IBC from IBC cells. This could be a potential diagnostic spectral marker for IBC detection useful for patient management.

In vitro vascularized tumor platform for modeling tumor-vasculature interactions of inflammatory breast cancer.

Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, microfluidic IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). Acellular collagen platforms with endothelialized blood vessels served as controls. SUM149 and MDA-MB-231 platforms exhibited a significantly (p < .05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of vascular endothelial growth factor (p < .05) and increased collagen ECM porosity compared to non-IBCMDA-MB-231 (p < .05) and control (p < .01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a key feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughput tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.

Heterogeneity at the invasion front of triple negative breast cancer cells

Identifying better predictive and prognostic biomarkers for the diagnosis and treatment of triple negative breast cancer (TNBC) is complicated by tumor heterogeneity ranging from responses to therapy, mutational burden, and clonal evolution. To overcome the gap in our understanding of tumor heterogeneity, we hypothesized that isolating and studying the gene expression profile of invasive tumor cell subpopulations would be a crucial step towards achieving this goal. In this report, we utilized a fluidic device previously reported to be capable of supporting long-term three-dimensional growth and invasion dynamics of cancer cells. Live invading and matched non-invading SUM149 inflammatory breast cancer cells were enriched using this device and these two functionally distinct subpopulations were tested for differences in gene expression using a gene expression microarray. 305 target genes were identified to have altered expression in the invading cells compared to the non-invading tumoroid cells. Gene ontology analysis of the gene panel identified multiple biological roles ranging from extracellular matrix reorganization to modulation of the immune response and Rho signaling. Interestingly, the genes associated with the invasion front differ between different samples, consistent with inter- and intra-tumor heterogeneity. This work suggests the impact of heterogeneity in biomarker discovery should be considered as cancer therapy increasingly heads towards a personalized approach.

Induction of heparanase via IL-10 correlates with a high infiltration of CD163+ M2-type tumor-associated macrophages in inflammatory breast carcinomas

Inflammatory breast cancer (IBC) is the most aggressive and lethal form of breast cancer, characterized by a high infiltration of tumor-associated macrophages and poor prognosis. To identify new biomarkers and to elucidate the molecular mechanisms underlying IBC pathogenesis, we investigated the expression pattern of heparanase (HPSE) and its activator cathepsin L (CTSL). First, we quantitated the HPSE and CTSL mRNA levels in a cohort of breast cancer patients after curative surgery (20 IBC and 20-non-IBC). We discovered that both HPSE and CTSL mRNA levels were significantly induced in IBC tissue vis-à-vis non-IBC patients (p <0 .05 and p <0 .001, respectively). According to the molecular subtypes, HPSE mRNA levels were significantly higher in carcinoma tissues of triple negative (TN)-IBC as compared to TN-non-IBC (p <0 .05). Mechanistically, we discovered that pharmacological inhibition of HPSE activity resulted in a significant reduction of invasiveness in the IBC SUM149 cell line. Moreover, siRNA-mediated HPSE knockdown significantly downregulated the expression of the metastasis-related gene MMP2 and the cancer stem cell marker CD44. We also found that IBC tumors revealed robust heparanase immune-reactivity and CD163+ M2-type tumor-associated macrophages, with a positive correlation of both markers. Moreover, the secretome of axillary tributaries blood IBC CD14+ monocytes and the cytokine IL-10 significantly upregulated HPSE mRNA and protein expression in SUM149 cells. Intriguingly, massively elevated IL-10 mRNA expression with a trend of positive correlation with HPSE mRNA expression was detected in carcinoma tissue of IBC. Our findings highlight a possible role played by CD14+ monocytes and CD163+ M2-type tumor-associated macrophages in regulating HPSE expression possibly via IL-10. Overall, we suggest that heparanase, cathepsin L and CD14+ monocytes-derived IL-10 may play an important role in the pathogenesis of IBC and their targeting could have therapeutic implications.

The immunomodulatory role of tumor Syndecan-1 (CD138) on ex vivo tumor microenvironmental CD4+ T cell polarization in inflammatory and non-inflammatory breast cancer patients.

Herein, we aimed to identify the immunomodulatory role of tumor Syndecan-1 (CD138) in the polarization of CD4+ T helper (Th) subsets isolated from the tumor microenvironment of inflammatory breast cancer (IBC) and non-IBC patients. Lymphocytes and mononuclear cells isolated from the axillary tributaries of non-IBC and IBC patients during modified radical mastectomy were either stimulated with the secretome as indirect co-culture or directly co-cultured with control and Syndecan-1-silenced SUM-149 IBC cells. In addition, peripheral blood mononuclear cells (PBMCs) of normal subjects were used for the direct co-culture. Employing flow cytometry, we analyzed the expression of the intracellular IFN-γ, IL-4, IL-17, and Foxp3 markers as readout for basal and co-cultured Th1, Th2, Th17, and Treg CD4+ subsets, respectively. Our data revealed that IBC displayed a lower basal frequency of Th1 and Th2 subsets than non-IBC. Syndecan-1-silenced SUM-149 cells significantly upregulated only Treg subset polarization of normal subjects relative to controls. However, Syndecan-1 silencing significantly enhanced the polarization of Th17 and Treg subsets of non-IBC under both direct and indirect conditions and induced only Th1 subset polarization under indirect conditions compared to control. Interestingly, qPCR revealed that there was a negative correlation between Syndecan-1 and each of IL-4, IL-17, and Foxp3 mRNA expression in carcinoma tissues of IBC and that the correlation was reversed in non-IBC. Mechanistically, Syndecan-1 knockdown in SUM-149 cells promoted Th17 cell expansion via upregulation of IL-23 and the Notch ligand DLL4. Overall, this study indicates a low frequency of the circulating antitumor Th1 subset in IBC and suggests that tumor Syndecan-1 silencing enhances ex vivo polarization of CD4+ Th17 and Treg cells of non-IBC, whereby Th17 polarization is possibly mediated via upregulation of IL-23 and DLL4. These findings suggest the immunoregulatory role of tumor Syndecan-1 expression in Th cell polarization that may have therapeutic implications for breast cancer.

Abstract P5-03-03: Polo-like Kinase 1 (PLK1) inhibition synergizes with docetaxel in basal breast cancer cells

Abstract Background. Triple negative breast cancer (TNBC) is a heterogeneous disease entity and gene expression analyses have identified molecular subtypes that are refining our understanding of breast cancer biology and enabling development of targeted therapy. The basal-like 2 (BL2) subtype, as described by Lehmann et al, is characterized by overexpression of EGFR, loss of PTEN, and mutations in the TP53 gene and those patients had a 0% pathologic complete response rate following neoadjuvant chemotherapy. Thus, BL2 breast cancers are intrinsically resistant to chemotherapy and patients with this type of breast cancer have a poor overall survival rate. In a recent genome-scale short hairpin RNA (shRNA) screen of breast cancer cells, PLK1 was a frequent and strong hit in the basal-like subtype, inflammatory breast cancer SUM149, indicating its importance for growth and survival of these breast cancer cells. PLK1 regulates progression of cells through the G2 phase of the cell cycle by phosphorylating FOXM1, which then regulates the expression of cyclins and other genes necessary for cells to progress through cell cycle. However, most of the drug companies discontinued clinical development of PLK inhibitors (PLK1i) (e.g. GSK461364 GlaxoSmithKline, Volasertib Boehringer Ingelheim) in solid tumors due to elevate toxicity shown in phase I-II studies. Methods. We selected GSK461364 as our PLKi for the in vitro experiments. GSK461364 was combined with docetaxel and cisplatin in a set of TNBC cells including SUM149, SUM159, SUM1315, SUM229, DU4475 and the control MCF10A. We performed cell proliferation assays in order to determine the IC25 (Inhibition Concentration of 25%) and IC50 concentration for each drug across the panel of cell lines. We then examined the influence of drugs on the clonogenic potential of the panel of cell lines, alone and in combination. Synergism or antagonism was determined using the Chou-Talalay method, by determining the combination index (CI) at Fa 0.5 (50% of cell death induced by drug treatment). We used FxCycle™ Stain (Invitrogen) for cell cycle analysis, and CD24/CD44 antibodies (BD Pharmingen™) for stem cell marker analysis. All experiments were carried in triplicate. Results. GSK461364 caused growth inhibition with IC50 varying from 6.1 nmol/L (SUM229) to 56.5 nmol/L (DU4475). In our growth assay, GSK461364 showed synergism with docetaxel in SUM149 (CI 0.70) and SUM159 (CI 0.62), and with cisplatin in SUM149 (CI 0.58), SUM159 (CI 0.85), and SUM229 (CI 0.54). Only GSK461364 in combination with docetaxel decreased the clonogenic potential of SUM149 (p &amp;lt; 0.001, Interaction Test, 0.001). In SUM149 cells, GSK461364 caused mitotic arrest in phase G2-M with the maximum effect 24 hours after administration of the drug. Moreover, when combined with docetaxel, GSK461364 reduced the relative percentage of CD44+/CD24-/dim population of SUM149. Conclusion. PLKi in combination with chemotherapy shows promising results in a subset of TNBC intrinsically resistant to chemotherapy. GSK461364 significantly decreased the clonogenic potential and stem cell population of SUM149 when combined to docetaxel. In combination with docetaxel, we demonstrated that is possible to lower the dose of PLKi by 5 folds in order to obtain the same activity. Citation Format: Giordano A, Liu Y, Armeson KE, Britten CD, Kappler CS, Ethier SP. Polo-like Kinase 1 (PLK1) inhibition synergizes with docetaxel in basal breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-03.

Biophysical Phenotyping and Modulation of ALDH+ Inflammatory Breast Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) have been shown to initiate tumorigenesis and cancer metastasis in many cancer types. Although identification of CSCs through specific marker expression helps define the CSC compartment, it does not directly provide information on how or why this cancer cell subpopulation is more metastatic or tumorigenic. In this study, the functional and biophysical characteristics of aggressive and lethal inflammatory breast cancer (IBC) CSCs at the single-cell level are comprehensively profiled using multiple microengineered tools. Distinct functional (cell migration, growth, adhesion, invasion and self-renewal) and biophysical (cell deformability, adhesion strength and contractility) properties of ALDH+ SUM149 IBC CSCs are found as compared to their ALDH- non-CSC counterpart, providing biophysical insights into why CSCs has an enhanced propensity to metastasize. It is further shown that the cellular biophysical phenotype can predict and determine IBC cells' tumorigenic ability. SUM149 and SUM159 IBC cells selected and modulated through biophysical attributes-adhesion and stiffness-show characteristics of CSCs in vitro and enhance tumorigenicity in in vivo murine models of primary tumor growth. Overall, the multiparametric cellular biophysical phenotyping and modulation of IBC CSCs yields a new understanding of IBC's metastatic properties and how they might develop and be targeted for therapeutic interventions.

Abstract 2397: Intracellular cholesterol regulates the DNA damage response in inflammatory breast cancer

Abstract Purpose: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer, and patients with IBC remain at high risk of locoregional recurrence after radiotherapy. We previously demonstrated that depleting intracellular cholesterol induces radiosensitivity in IBC cells in vitro, and that IBC patients with high levels of high-density lipoprotein (HDL) and those taking a statin, which inhibits de novo cholesterol biosynthesis, have improved locoregional control after radiotherapy. These results suggest that targeting cholesterol metabolism may improve the radiotherapeutic management of IBC. Here, we aimed to understand the molecular mechanism(s) linking cholesterol metabolism and radiation-induced DNA damage and DNA repair and to evaluate statin pharmacotherapy as a DNA repair inhibitor and radiotherapeutic adjunct in an in vivo pre-clinical model of IBC. Methods: KPL4, SUM149, SUM190, and IBC3 IBC cell lines were used to examine the effect(s) of simvastatin and human serum lipoproteins on radiation-induced DNA damage induction, DNA damage signaling, DNA damage-associated G1, intra-S, and G2/M cell cycle checkpoint activation, and DNA double strand break (DSB) repair by homologous recombination (HR) and non-homologous end joining (NHEJ). Results: We have generated multiple IBC cells lines with an integrated Traffic Light Reporter (TLR) that is capable of measuring HR and NHEJ, and demonstrate that depletion of intracellular cholesterol abrogates HR-mediated repair of DNA DSBs. We also show that intracellular cholesterol has pleiotropic effects on cell cycle distribution and DNA damage-associated cell cycle checkpoints. We also report a novel polycistronic dual bioluminescence reporter system that can robustly visualize and quantitate DNA DSB induction and repair in vitro and in vivo. Conclusions: Cholesterol is an important determinant of intrinsic radiosensitivity in IBC, and appears to regulate repair of radiation-induced DNA DSBs. Targeting cholesterol metabolism is a potential strategy to overcome IBC radioresistance and improve the prognosis for this aggressive disease. Citation Format: Shane R. Stecklein, Adam R. Wolfe, Bisrat G. Debeb, Richard A. Larson, Wendy A. Woodward. Intracellular cholesterol regulates the DNA damage response in inflammatory breast cancer [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 2397.

CSF-1/CSF-1R axis is associated with epithelial/mesenchymal hybrid phenotype in epithelial-like inflammatory breast cancer

Inflammatory breast cancer (IBC) is a rare subtype of breast cancer, accounting for 8–10% of breast cancer-associated deaths in the US. Clinical hallmarks of IBC include tumor emboli in lymphatic vessels and E-cadherin overexpression, which supports a type of metastasis referred to as cell cluster-based metastasis, prevalent in IBC. In contrast, we previously reported epithelial-to-mesenchymal transition (EMT)-based progression of IBC, utilizing in vivo xenografts and in vitro Matrigel culture models. To address these two contradictory concepts of IBC metastasis, we used Matrigel culture to induce EMT in a panel of IBC cells. Results revealed Matrigel culture induced vimentin expression in SUM149 and SUM190 IBC cells at the transcriptional and protein levels while maintaining the expression of E-cadherin, a phenomenon referred to as partial EMT. Transcriptional profiling revealed that expression of colony-stimulating factor 1 (CSF-1) was induced in Matrigel culture. When the receptor tyrosine kinase of CSF-1 (CSF-1R) was inhibited by CSF-1R inhibitor BLZ945, the partial EMT was reversed in a dose-dependent manner, indicating that the CSF-1/CSF-1R axis plays a key role in controlling partial EMT. This observation may help reconcile the two contradictory theories of IBC metastasis, EMT vs cell cluster-based metastasis.

Expression Pattern of Hyaluronan Synthase2 in Inflammatory Breast Cancer Cells

Breast cancer (BC) Is the most widely recognized threatening malignant disease around the whole world. It Is the second most incessant reason for malignant tumor growth related to death among woman at different ages. Inflammatory Breast Cancer (IBC) Is the most lethal form of BC with low survival rate. Because of lacking molecular markers, identification of new unique markers for IBC Is warranted. Hyaluronic acid synthases (HASs), particularly HAS2, have shown a critical role in the metastasis of non-inflammatory breast cancer (non-IBC). In this study, we assessed HAS2 mRNA expression by qPCR in the human triple negative non-IBC MDA-MB-231 and IBC SUM-149 cell lines. Our data demonstrated that HAS2 mRNA expression was significantly upregulated in IBC vs. non-IBC breast cancer cell lines. In conclusion, the high expression of HAS2 may represents a marker for IBC and could have a role in IBC pathogenesis.

Abstract PD6-06: Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients

Abstract Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [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 PD6-06.

Experimental study on inhibiting invasion of inflammatory breast cancer cell lines SUM149 and SUM190 by silencing RhoC gene with the help of RNA interference

Objective In this study, we silence RhoC gene in inflammatory breast cancer (IBC) SUM149, SUM190 cell lines and explore its effects on cell invasion of IBC by RNA interference (RNAi) technology so as to provide experimental principal for target gene therapy of IBC. Methods Human IBC cells were divided blank group, control siRNA group, RhoC-siRNA group by balance randomization. RhoC-siRNA was used to transfect human IBC SUM149, SUM190 cell lines. Microscopy was applied to analyze transfection efficiency. After transfecting for 48 hours, reverse transcriptase-polymerase chain reaction (RT-PCR) was made to detect mRNA expression levels of RhoC. Western blotting was also used to analyze the expression level of RhoC protein. After transfecting for 48 hours, the ability to invasion was analyzed by transwell hole. Results We used siRNA to transfect human IBC SUM149, SUM190 cell lines and found that transfection efficiency can exceed to 80%. After we transfected for 48 hours, the expression levels of RhoC protein in IBC SUM149 cell lines 0.375±0.164 in RhoC siRNA group were significantly lower than those 0.897±0.376 in control siRNA group (t=6.274, P=0.045). The expression levels of RhoC protein in IBC SUM190 cell lines 0.381±0.166 in RhoC siRNA group were significantly lower than those 0.902±0.386 in control siRNA group (t=6.891, P=0.028). However, there were no obvious differences between in blank group and control siRNA group. After we transfected for 48 hours, in IBC SUM149 cell lines, the ability to invasion 85.6±12.7 in RhoC siRNA group was markedly lower than that 340.5±43.4 in control siRNA group (t=12.275, P=0.034). In IBC SUM190 cell lines, the ability to invasion 92.8±15.6 in RhoC siRNA group was markedly lower than that 345.9±47.7 in control siRNA group (t=11.769, P=0.022). Conclusion RhoC is known as an important role in the carcinogenesis of IBC. With the help of RNAi technology, we can greatly inhibit the invasion of IBC SUM149, SUM190 cell lines by silencing RhoC gene. RNAi technology perhaps becomes an important gene therapy tool for IBC. Key words: Inflammatory breast cancer; RhoC; RNA interference; Gene silence

Overexpression of caveolin-1 in inflammatory breast cancer cells enables IBC-specific gene delivery and prodrug conversion using histone-targeted polyplexes.

Gene therapy platforms offer a variety of potentially effective solutions for development of targeted agents that can be exploited for cancer treatment. The physicochemical properties of nanocarriers can be tuned to enhance their localization in tumors, and cell specificity can also be increased by appropriate selection of gene targets. A relatively underexploited approach to enhance therapeutic selectivity in cancer tissues is the use of nanocarriers whose nuclear targeting and uptake are triggered by the altered expression of specific endomembrane trafficking proteins in cancer cells. Previously, we showed that histone 3 (H3) peptide-targeted DNA polyplexes traffic to the nucleus efficiently through caveolar endocytosis followed by transfer through the Golgi and endoplasmic reticulum (ER). We hypothesized that these polyplexes would exhibit enhanced activity in inflammatory breast cancer (IBC) cells, which overexpress caveolin-1 as part of their invasive phenotype, and we also posited that this targeting effect could be exploited to facilitate IBC-specific transfection and prodrug conversion in the presence of normal breast epithelial cells. Using cellular transfection experiments, function-blocking assays, and confocal imaging in both IBC SUM149 cell monocultures and IBC SUM149 co-cultures with MCF10A normal breast epithelial cells, we found that our H3-targeted polyplexes selectively transfected IBC SUM149 cells at a 4-fold higher level than normal breast epithelial cells. This selectivity and increased transfection were caused by a 2.2-fold overexpression of caveolin-1 in IBC SUM149 cells, which led to increased polyplex trafficking to the nucleus through the Golgi and ER. We also saw similar enhancements in cell selectivity and transfection when cells were transfected with a suicide gene/prodrug combination, as the increased expression of the suicide gene in IBC SUM149 cells led to a 55% decrease in viability in IBC SUM149 cells as compared to a 25% decrease in MCF10A cells. These findings demonstrate that differences in the expression of the endocytic membrane protein caveolin-1 can be exploited for cell-selective gene delivery, and ultimately, these gene-based targeting approaches may be useful in potential treatments for aggressive cancer types. Biotechnol. Bioeng. 2016;113: 2686-2697. © 2016 Wiley Periodicals, Inc.