Inflammatory Breast Cancer Cells 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.

Abstract PO2-14-10: A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model

Abstract Background Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses but up to 30% of breast cancer-related deaths. Similarly, Inflammatory breast cancer (IBC) accounts for 2-4% of breast cancer diagnosis but causes 8%–10% of all breast cancer-related deaths. The myeloid cell leukemia-1 (MCL-1) is a member of the BCL-2 family of proteins, which is highly amplified in numerous human cancers and associated with cell immortalization, transformation, and chemoresistance. In fact, MCL-1 is amplified in 55% of TNBC after preoperative chemotherapy. Amongst the MCL-1 inhibitors that have been developed, AZD5991 showed high selectivity and affinity for MCL-1. In myeloma and acute myeloid leukemia cells AZD5991 induces apoptosis and showed anti-tumor effects in in vivo models. In the present study, we evaluated the biological role of the MCL-1 inhibitor AZD5991 in IBC and TNBC in vitro and in vivo models. Materials and methods The expression of the MCL-1 gene was analyzed in the World Consortium IBC patient dataset. Next, we screened a panel of TNBC and IBC cell lines for MCL-1 expression by western blot analysis. The effect of AZD5991 on TNBC and IBC cells was analyzed via cell titer-blue proliferation, clonogenic assays and soft agar assay. To further validate these findings, we used siRNA to knockdown MCL-1 and performed cell proliferation, clonogenic and migration assays. To identify signaling pathways involved in MCL-1 Inhibitor’s effects we performed a proteome profiler human apoptosis array in TNBC cells in the presence of AZD5991. The anti-tumor effect of the MCL-1 inhibitor was evaluated using a SUM-149 (TN-IBC) orthotopic xenograft mouse model. The mice were treated with AZD5991 at doses of 15 and 30mg/kg once weekly via tail vein injection for a duration of 4 weeks. Results The mean expression level of MCL-1 was higher in patients with TNBC than in those with non-TNBC. Further, compared to the patients with ER positive, HER-2 negative tumors those with TNBC showed an increased in the mean expression of MCL-1 gene (p value= 0.05). We screened a panel of TNBC and IBC cell lines in which most of the cell lines showed high levels of MCL-1. The cell proliferation assay showed IC50 ranging from 0.27 – 19.6 μM. The SUM149, SUM190, FCIBC02, MDA-IBC3 and MDA-MB-468 cells showed high sensitivity and showed significant decrease in the proliferation rate while BCX010, KPL4, MDA-MB-231, 4T1.2 and E0771 cells as well as the normal breast cells MCF10A exhibited resistance with no significant decrease in proliferation. The clonogenic and soft agar assays with these sensitive and resistant cell lines showed similar results. Further, these findings were validated via genetic knockdown of MCL-1 which resulted in a significant decrease in cell proliferation, colony formation and migration capability of the TNBC cells. To identify signaling pathways involved in MCL-1 Inhibitor’s phenotypic changes, we performed a proteome profiler human apoptosis array which showed a decreased in BCLx expression, and an increase in expression of BAX and phospho-Rad17 proteins in the AZD5991 treated TN-IBC cells. Finally, in a SUM-149 (TN-IBC) orthotopic xenograft mouse model, mice injected with 30mg/kg showed a significant tumor regression compared to the vehicle-treated mice (vehicle vs 30 mg/kg, p value=0.0006). Conclusions and Future directions Our data demonstrates that AZD5991 inhibits tumorigenesis in an inflammatory breast cancer xenograft model. We are currently validating several significant targets to understand the biological mechanisms related to our candidate drug. Our long-term goal is to develop MCL-1 targeted therapy in combination with standard-of-care treatment for inflammatory and triple-negative breast cancers. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Michael White, Bisrat Debeb, Emilly Villodre, Xiaoding Hu, Debu Tripathy, Wendy Woodward, Rachel Layman, Geoffrey Bartholomeusz, Chandra Bartholomeusz. A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model [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 PO2-14-10.

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [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 PO2-24-06.

Abstract 2100: Novel LIPA targeted therapy for treating inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare but incredibly aggressive subtype of breast cancer (BC). IBC accounts for 2-4% of all occurrences of breast cancer and results in 7-10% of breast cancer-related deaths. IBC is only partially treatable with current therapies such as chemotherapy, surgery, and radiotherapy. Identification of novel therapeutic targets is urgently required. The main organelle for the synthesis, folding, and modification of proteins is called the endoplasmic reticulum (ER). Since elevated basal ER stress (ERS) is usually found in many cancers including IBC, our hypothesis was that the high basal ERS in IBC constitutes a serious vulnerability that can be targeted. Recently, we developed a small molecule ERX41 that promotes ER stress by blocking Lysosomal acid lipase A (LIPA) function in ER of cancer cells. The goal of the project is to test the utility of ERX-41 in treating IBC. Methods: To study the effect of ERX-41, we have used three well-established IBC model cells. We evaluated the efficacy of ERX-41 as a novel therapeutic for treating IBC using cell viability assays. The long-term effects of ERX-41 on IBC cell survival were evaluated using colony formation assays. Annexin V assays were used to measure apoptosis. Reporter assays, splicing assays, RT-qPCR, and Western blotting were used in mechanistic investigations. The effectiveness of ERX-41 was examined in vivo using KPL4 cell-based xenografts. Results: ERX-41 significantly decreased the viability of all three IBC model cells (SUM149, SUM190PT, and KPL4), with an IC50 of about 125 nM in MTT assay. Additionally, ERX-41 treatment significantly decreased IBC cells capacity to form colonies and promoted apoptosis. Specificity of the ERX41 mediated actions were confirmed using LIPA KO cells. Using RTqPCR based splicing assays, we demonstrated increased splicing of XBP1 as early as 6 hours after ERX41 treatment. Western analyses showed robust induction of stress markers (CHOP, PERK, ATF4) in IBC cells upon treatment with ERX-41. In KPL4 xenograft studies, ERX-41 showed significant reduction in the tumor volume. IHC analyses confirmed decreased proliferation marker and increased ER stress markers. Conclusions: Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of action and implicate ERX-41 binding to LIPA induces ER stress and promotes apoptosis of IBC cells. Citation Format: Zenaida Fuentes, Rahul Gopalam, Bianca A. Romo, Khaled Mohamed Nassar, Xue Yang, Behnam Ebrahimi, Paulina Ramirez, Chia-Yuan Chen, Scott Elmore, Harika Nagandla, Uday Pratap, Christoforos Thomas, Suryavathi Viswanadhapalli, Jung-Mo Ahn, Ganesh Raj, Ratna K. Vadlamudi. Novel LIPA targeted therapy for treating inflammatory breast cancer [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 2100.

Abstract 5986: Significance of LIF/LIFR axis in the progression of inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare form of locally progressed breast cancer. Even though it is uncommon, IBC accounts for 7% of breast cancer deaths. There is a critical need for novel therapeutic targets to develop new therapeutics. IBC is more common in young women. Altered levels of growth factors, and cytokine signaling are suspected to play a role in the progression of IBC. Leukemia inhibitory factor (LIF) is the most pleiotropic member of the interleukin-6 family of cytokines. LIF and its receptor LIFR have been linked to the progression of many cancers including triple negative breast cancer. We recently developed a first-in-class inhibitor targeting LIFR named EC359. However, the role of the LIF/LIFR axis in IBC progression remains unknown. In this study, we examined the significance of LIFR and the effectiveness of LIFR inhibitor EC359 in treating IBC. Methods: The effect of LIFR inhibitor EC359 on cell survival, colony formation, and apoptosis was evaluated using three well-established IBC model cells (SUM149, SUM190PT, and KPL4). Mechanistic studies were conducted using Western blotting, CRISPR KO, reporter assays, and RT-qPCR. KPL4 cell-based xenografts were used to test the efficacy of EC359 in vivo. Results: Our results using Western blotting confirmed increased expression of LIF and LIFR in IBC cells compared to normal and ER+ breast cancer cells. Treatment with EC359 significantly decreased IBC cell viability, long term colony formation ability with an IC50 of ~10-20 nM. Similarly, knock out of LIFR significantly reduced the progression of IBC model cells in cell viability and colony formation assays. Further EC359 treatment promoted apoptosis of IBC cells. Mechanistic studies confirmed that EC359 treatment substantially reduced LIF/LIFR downstream signaling including STAT3, AKT, and mTOR signaling. We also tested the utility of EC359 in treating IBC in vivo using KPL4 xenografts. EC359 (5mg/kg/day) is highly efficacious in reducing IBC xenograft tumor growth in vivo. IHC analyses of tumors confirmed decreased cell proliferation as measured by Ki67 staining. Conclusion: Collectively, these studies demonstrated the therapeutic benefit of using EC359 to target the LIF/LIFR axis and opened the door for the development of new treatment approaches. Citation Format: Bianca Romo, Zenaida Fuentes, Lois Randolph, Bindu Santhamma, Hareesh Nair, Christoforos Thomas, Ratna Vadlamudi, Suryavathi Viswanadhapalli. Significance of LIF/LIFR axis in the progression of inflammatory breast cancer [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 5986.

Abstract 4482: Development of small molecule inhibitors of lipocalin 2 for the treatment of aggressive cancers

Abstract Lipocalin-2 (LCN2), a secreted protein pivotal in iron homeostasis, immune responses, siderophore transport, and epithelial cell differentiation. LCN2 is highly abundant in aggressive forms of many cancers including breast, pancreatic, ovarian, and esophageal cancer. Elevated LCN2 levels in aggressive tumors correlate with heightened cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. After the protein folds it makes a barrel structure which encloses a three-calyx pocket that can bind and transport molecules. A first generation of small molecule inhibitors (SMIs) targeting LCN2 demonstrated efficacy in attenuating the proliferative capacity of Inflammatory breast cancer (IBC) cells. IBC stands as a rare and highly aggressive variant, accounting for 1 to 5% of all BC cases, with 20-30% presenting as de novo metastatic disease. In the previous studies the concentration of LCN2 inhibitors to reduce cell proliferation in 50% was around 10 µM. In pursuit of better LCN2 SMIs, we conducted in-silico analysis using a library of 369,000 ligands. Through the High-Performance Computer Facility at UPR Río Piedras, we employed AutoDock Vina for virtual screening, targeting the LCN2-calyx with Lys134 as the central coordinate. Docking protocols, predicting binding affinities, identified 8,162 out of 327,638 ligands that have been docked already with values below -9.6 kcal/mol for further consideration. The next phase involves subjecting these potential inhibitors to the SwissADME tool for a comprehensive evaluation of absorption, distribution, metabolism, and excretion parameters, as well as pharmacokinetics, drug-likeness, and medicinal chemistry considerations. The parameters to be evaluated are the Lipinski rules, which states that an orally active drug may have a maximum of one violation and the pann-assay interference compounds (PAINS) alerts tool, a source of false positives for biologically active compounds. The top candidates, exhibit binding affinities within the range of -11.5 to -12.2. After SwissADME filtering, we will re-rank the ligand list, identifying 30 commercially available candidates for subsequent cell viability assays in SUM149 (TNBC) and SUM190 (HER2+) cell lines. This multifaceted approach integrates computational methodologies with rigorous experimental validation, paving the way for potential therapeutic interventions in IBC treatment. Citation Format: Marienid Flores-Colón, Cristal A. Peña-Vázquez, Eliud Hernández-O'Farril, Luis E. Vázquez-Quiñones, Pablo E. Vivas-Mejía. Development of small molecule inhibitors of lipocalin 2 for the treatment of aggressive cancers [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 4482.

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.

Influence of Macrophages on Vascular Invasion of Inflammatory Breast Cancer Emboli Measured Using an In Vitro Microfluidic Multi-Cellular Platform.

Inflammatory breast cancer (IBC) is an aggressive disease with a poor prognosis and a lack of effective treatments. It is widely established that understanding the interactions between tumor-associated macrophages (TAMs) and the tumor microenvironment is essential for identifying distinct targeting markers that help with prognosis and subsequent development of effective treatments. In this study, we present a 3D in vitro microfluidic IBC platform consisting of THP1 M0, M1, or M2 macrophages, IBC cells, and endothelial cells. The platform comprises a collagen matrix that includes an endothelialized vessel, creating a physiologically relevant environment for cellular interactions. Through the utilization of this platform, it was discovered that the inclusion of tumor-associated macrophages (TAMs) led to an increase in the formation of new blood vessel sprouts and enhanced permeability of the endothelium, regardless of the macrophage phenotype. Interestingly, the platforms containing THP-1 M1 or M2 macrophages exhibited significantly greater porosity in the collagen extracellular matrix (ECM) compared to the platforms containing THP-1 M0 and the MDA-IBC3 cells alone. Cytokine analysis revealed that IL-8 and MMP9 showed selective increases when macrophages were cultured in the platforms. Notably, intravasation of tumor cells into the vessels was observed exclusively in the platform containing MDA-IBC3 and M0 macrophages.

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.

Engineering E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells.

Naturally occurring protein nanocages are promising drug carriers as both the interior and exterior can be decorated for drug encapsulation and cell targeting. To provide surface functionalization, we added a SpyTag to E2 nanocages (ST-E2) to enable tunable decoration using the robust SpyCatcher bioconjugation strategy. Additionally, the E2 core was mutated with four phenylalanine substitutions for doxorubicin loading and pH-responsive release. By decorating the exterior with a highly cell-specific epidermal growth factor receptor (EGFR)-targeting protein conjugate, 4GE11-mCherry-SpyCatcher, we demonstrated targeted cell death in inflammatory breast cancer cells compared to healthy breast epithelial cells at concentrations below the IC50 of free doxorubicin.

Engineering Hepatitis B Virus (HBV) Protein Particles for Therapeutic Delivery.

Nature provides an abundance of proteins whose structures and reactivity have been perfected through evolution to perform specific tasks necessary for biological function. The structural and functional properties of many natural proteins are quite valuable for the construction and customization of drug delivery vehicles. Self-assembling protein nanoparticle platforms are particularly useful scaffolds, as their multi-subunit designs allow the attachment of a high density of modifying molecules such as cell-binding ligands that provide avidity for targeting and facilitate encapsulation of large quantities of therapeutic payload. We explored SpyCatcher/SpyTag conjugation as a system to modify hepatitis B virus (HBV)-like particles (HBV VLPs). Using this simple decoration strategy, we demonstrated efficient and cell-selective killing of inflammatory breast cancer cells via delivery of yeast cytosine deaminase suicide enzymes combined with 5-fluoro-cytosine prodrugs.

A study of antibody conjugates for the treatment of inflammatory breast cancer.

e15016 Background: Inflammatory breast cancer (inflammatory carcinoma) is a rare metastatic malignant tumor, it accounts for about 2% of all breast cancer cases. Despite its low incidence, inflammatory breast cancer is highly invasive, its five-year survival rate is less than 41%, making its prognosis the worst of all breast cancer subtypes, and effective drugs are urgently needed in clinical practice. Therefore, our study develops novel antibody conjugate drugs that can target overexpressed-ICAM-1 in inflammatory breast cancer cells as an original molecular target precisely. Methods: In this study, flow cytometry was used to perform precise and quantitative target screening of inflammatory breast cancer cell lines (SUM-149, KPL4), the endocytosis efficiency of ADC drugs was verified by flow cytometry efficiency analysis and single-photon confocal analysis, and the cytotoxicity of different drugs and cell lines was explored by CCK8 experiment. A nude mouse model of orthotopic xenograft inflammatory breast cancer was constructed, and the in vivo therapeutic effect of inflammatory breast cancer was observed by intravenous injection of two ICAM-1 antibody conjugates (ICAM-1-MMAE and ICAM-1-DXD), also the ADC drug structure with the best therapeutic effect for inflammatory breast cancer was screened. Results: ICAM-1 molecule in a variety of inflammatory breast cancer cell lines were highly over-expressed, and the antibody internalization rate was 51% at 4h, and after the peak the endocytosis efficiency slowed down into the plateau stage; ICAM1-MMAE and ICAM1-DXD are safer than first-line chemotherapy agents for breast cancer; The tumor volume was significantly reduced after administration into the orthotopic breast cancer xenograft model, which further verified the obvious effect of ADC drugs in vivo. Conclusions: Both ICAM-1-MMAE and ICAM-1-DXD have shown significant anti-inflammatory breast cancer effects in vitro and in vivo, and this study provides potential antibody conjugate drugs for targeted therapy of inflammatory breast cancer, which is expected to help improve the poor prognosis of inflammatory breast cancer.

Abstract 1580: LCN2 is a therapeutic target against inflammatory breast cancer

Abstract Breast cancer (BC) is the most common cancer type accounting for 12% of annual cancer cases worldwide. In the United States, 30% of the newly diagnosed cancer cases in woman are BC. Inflammatory breast cancer (IBC) is a rare and aggressive subtype of BC that accounts for 1 to 5% of all types of BC. Due to its inflammatory characteristics and the absence of a palpable mass, IBC is usually confounded with a mastitis. Once properly diagnosed, IBC has already metastasized. A high portion of IBC tumors overexpress human epidermal growth factor receptor 2 (HER2). These IBC patients (HER2+) are treated with Trastuzumab, a monoclonal antibody. However, many IBCs become resistant to this therapy. Thus, there is not an optimal treatment against this horrendous disease. One potential target against IBC is Lipocalin-2 (LCN2), a secreted protein involved in iron homeostasis, immune responses, transport of siderophores, and epithelial cell differentiation. In many aggressive tumors, high levels of LCN2 have been associated with increased cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. Our research team published that LCN2 is aberrantly abundant in IBCs compared with non-IBC patients and cell lines and showed that small interference RNAs (siRNAs) targeting LCN2 effectively reduced the cell proliferation and invasion ability of IBC cells. Therefore, to understand the mechanisms involved after LCN2 knockdown, we did a RNA-sequencing using IBC3 cells transfected with LCN2-siRNA or with a negative control siRNA, where we identified 138 dysregulated genes. Genes as SOX18, which correlates to distant metastasis and poor clinical prognosis in cancer, and as LRP4, which correlates to proliferating clinical features by activating the PI3K/AKT pathway were identified. We used the Ingenuity Pathway Analysis software to identify the pathways regulated after LCN2 knockdown and found 25 canonical pathways altered. These results will be further validated by qPCR and Western Blot analysis. Also, after the identification of LCN2 as a plausible target for therapy against IBC, we worked on the preparation of a Herceptin-conjugated liposome loaded with siRNA against LCN2, to improve internalization in HER2+ IBC cells. We developed a three step reaction to obtain trastuzumab DSPE-PEG(2000) maleimide conjugates used to prepare the liposomal formulation. Trastuzumab conjugated liposomes were prepared and characterized using dynamic light scattering to measure their size distribution. Our formulation displayed sizes of 18.3 and 73.7 nm which could correspond to free DSPE-PEG(2000) trastuzumab and the trastuzumab-liposome, respectively. There is ongoing work to determine the optimal timepoint for internalization of liposomes into IBC cell lines and further treatment with liposomes charged with siRNA LCN2 to treat cell lines and determine the biological effect with the aim of developing a targeted treatment for inflammatory breast cancer. Citation Format: Marienid Flores-Colon, Pablo E. Vivas-Mejia. LCN2 is a therapeutic target against inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1580.

Sensitization of Resistant Cells with a BET Bromodomain Inhibitor in a Cell Culture Model of Deep Intrinsic Resistance in Breast Cancer

We treated highly metabolically adaptable (SUM149-MA) triple-negative inflammatory breast cancer cells and their control parental SUM149-Luc cell line with JQ1 for long periods to determine its efficacy at inhibiting therapy-resistant cells. After 20 days of treatment with 1-2 µM of JQ1, which killed majority of cells in the parental cell line, a large number of SUM149-MA cells survived, consistent with their pan-resistant nature. Interestingly, though, the JQ1 treatment sensitized resistant cancer cells in both the SUM149-MA and SUM149-Luc cell lines to subsequent treatment with doxorubicin and paclitaxel. To measure JQ1-mediated sensitization of resistant cancer cells, we first eradicated approximately 99% of relatively chemotherapy-sensitive cancer cells in culture dishes by long treatments with doxorubicin or paclitaxel, and then analyzed the remaining resistant cells for survival and growth into colonies. In addition, combination, rather than sequential, treatment with JQ1 and doxorubicin was also effective in overcoming resistance. Notably, Western blotting showed that JQ1-treated cancer cells had significantly lower levels of PD-L1 protein than did untreated cells, indicating that JQ1 treatment may reduce tumor-mediated immune suppression and improve the response to immunotherapy targeting PD-L1. Finally, JQ1 treatment with a low 62.5 nM dose sensitized another resistant cell line, FC-IBC02-MA, to treatment with doxorubicin and paclitaxel.

Abstract P6-10-08: Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers

Abstract Background Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are aggressive breast cancer subtypes with poor clinical outcomes due to the lack of well-validated and actionable targets and the onset of chemo-resistant metastasis. TNBC and IBC account for 15-20% and 2-4% of all breast cancer diagnoses but result in 30% and 8%–10% of breast cancer-related deaths, respectively. MELK (maternal embryonic leucine zipper kinase) is a potential therapeutic target in both TNBC and triple-negative IBC (TN-IBC). We have shown that 1) MELK expression is higher in basal tumors (mostly TNBC) and in IBC than in non-IBC, 2) high MELK expression is an independent prognostic factor for poor overall survival (P=0.0002) and poor distant metastasis-free survival (P=0.008) in breast cancer, and 3) MELK knockout leads to significantly lower metastatic burden and prolonged survival in a xenograft model of TNBC (unpublished data). MELK inhibitor (MELKi) OTS167 is in clinical trials but it lacks specificity and cross-reacts with other essential kinases. It is critical to discover novel, selective inhibitors with good bioavailability that target MELK and minimize adverse effects. We have developed a second-generation small-molecule MELK inhibitor 30e that is a highly potent and slow-binding ATP-competitive inhibitor of MELK. 30e exhibits potent cellular inhibition of MELK (IC50&amp;lt; 10 nM), as assessed using a live cell NanoBRET assay, and is highly selective as assessed by live-cell KiNativ profiling experiments in MDA-MB-231 TNBC cells. Methods We screened a panel of TNBC and IBC cell lines for MELK expression by western blot and qPCR analysis. We assessed the effects of MELK inhibition with the 30e on migration and invasion. The role of MELK in regulating cancer stem cell phenotypes was assessed using the mammosphere assay and flow cytometry (CD44+/CD24-/low surface marker). MELK’s protumorigenic role was determined in vitro using a soft agar assay, and the anti-tumor activity of 30e was assessed in vivo using a TN-IBC orthotopic xenograft mouse model. Results We found that TNBC had high mRNA levels of MELK (62%) compared to non-TNBC cell lines (25%), which correlated with MELK protein levels. 30e inhibited cell viability in TNBC and IBC cells in a dose-dependent manner and with IC50s ranging from 0.45 to 1.76 μM (P≤0.05). However, no significant effect was observed when normal MCF-10A breast cells were treated with 30e (IC50&amp;gt;20 μM). 30e also significantly reduced colony formation ability. Further, we observed that 30e reduced mammosphere formation efficiency and the CD44+/CD24-/low subpopulation in both TNBC and IBC cells in a dose-dependent manner (P≤0.05), suggesting the potential of 30e to inhibit the stem cell population in vitro. The soft agar assay also showed a significant decrease in colony formation in the TNBC and IBC cells after treatment with 30e, an indirect indicator of in vivo tumorigenicity. To assess the efficacy of 30e treatment on tumor growth, we evaluated this inhibitor as a single agent in a SUM-149 (TN-IBC) orthotopic mouse model. The mice were treated with intraperitoneal injections daily with 3 doses (2.5, 5, and 10 mg/kg) of 30e. Our data show that 30e suppressed tumor growth in a dose-dependent manner (P&amp;lt; 0.007). Conclusions and Future Directions Our data demonstrate that a novel, potent and specific MELK inhibitor inhibits tumor growth by suppressing the cancer stem cell phenotype. MELK is a promising target for aggressive cancers such as TNBC and IBC. We are evaluating how MELK inhibition modulates the tumor microenvironment, which is a critical component of breast cancer response to treatment. We performed a cytokine antibody array and are validating the top targets. Further, we will evaluate 30e in combination with standard-of-care treatment for toxicity and inhibition of metastasis. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Hector Gonzalez, Emilly S. Villodre, Bisrat Debeb, Debu Tripathy, Geoffrey Bartholomeusz, Juhyeon Lee, Kevin Dalby, Chandra Bartholomeusz. Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-08.

Abstract GS5-08: Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer with rapid onset and a strong propensity to spread to distant organs. Five-year overall survival (OS) rates remain poor, in part because of the high risk of brain metastasis: 19% of patients with IBC have brain metastases within the first 2 years after diagnosis. Our hypothesis for this study was that soluble E-cadherin (sEcad), an 80-kDa extracellular proteolytic fragment of full-length E-cadherin - a tumor promoter in IBC, is crucial for driving brain metastasis in IBC. Methods: We analyzed serum sEcad levels in from 348 IBC patients by ELISA. Four IBC cell lines [ER–/HER2+ (MDA-IBC3; SUM190) and ER–/HER2– (SUM149; BCX010)], human brain microvascular endothelial cells, and immortalized human astrocytes were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Mass spectrometry and Bio-ID-based proteomics assays, and RNA sequencing were used to identify sEcad-interacting proteins and potential mechanisms. In vivo, we studied tumor growth and brain metastasis in mice by injecting IBC cells into the mammary fatpad or tail vein, respectively, of SCID/Beige mice. Results: In IBC patients, higher serum sEcad levels correlated with poorer OS (p=0.02), earlier development of metastasis (p=0.006), and development of brain metastasis (p=0.04). On multivariable analysis, sEcad independently predicted OS (hazard ratio [HR]=2.07 [95% CI 1.19-3.60], p=0.01). In vitro, sEcad overexpression in IBC cell lines promoted anchorage-independent growth, migration, invasion, and resistance to anoikis. In vivo, sEcad-overexpressing SUM149 cells promoted primary tumor growth (p=0.007). Mice injected with sEcad-overexpressing MDA-IBC3 cells also had higher incidence of brain metastasis (100% vs 50%, p=0.03), metastatic burden (p=0.02) and number of metastases per mouse (p=0.0009), and had worse OS (p=0.0016), and brain metastasis-free survival (p=0.04), relative to controls. We further found that sEcad increased cancer cell adhesion to brain endothelial cells (p=0.01) and promoted induction of reactive astrocytes (as identified by high glial fibrillary acidic protein levels) in vitro and in vivo. Mechanistically, mass spectrometry and Bio-ID assays identified X-linked inhibitor of apoptosis protein (XIAP), a potent inhibitor of apoptotic cell death, as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Further analysis showed that sEcad bound to the BIR2 domain of XIAP. XIAP is the most potent and best-defined anti-apoptotic IAP family member, and it could induce NF-κB activation to inhibit tumor cell apoptosis Gene set enrichment analysis of RNA-seq profiling data showed activation of NF-kB signaling and downregulation of apoptotic pathways in the sEcad-overexpressing SUM149 cells compared with controls. Immunoblotting revealed that sEcad enhanced XIAP expression, activated NF-κB signaling, and inhibited cleavage of caspase-3 in IBC cells. Conclusions: We found that higher serum sEcad correlates with development of brain metastases and independently predicts poor OS in patients with IBC. We further found that sEcad promotes tumor growth and brain metastasis, perhaps via activation of XIAP/NF-κB signaling in breast cancer cells and promotion of endothelial cell adhesion and reactive astrocytosis in the brain microenvironment. These findings uncover a novel and crucial role for sEcad in brain metastasis and provide new insights and potential therapeutic targets for patients with metastatic IBC. Citation Format: Xiaoding Hu, Yun Xiong, Emilly S. Villodre, Juhee Song, Ganiraju C Manyam, Moises J Tacam, Jing Wang, Chandra Bartholomeusz, Debu Tripathy, Naoto T. Ueno, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-08.

Abstract P6-10-15: Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity

Abstract Background. Because of its high metastatic potential, inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer. We previously demonstrated that Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein overexpression was associated with shorter metastasis-free survival (MFS) in IBC patients, but not in non-IBC (nIBC) patients. However, the mechanism of action of MARCKS and its particular association to poorer outcome in IBC compared to nIBC are poorly understood. Methods. We evaluated in vitro the inhibitory effect of MPS (MARCKS phosphorylation site domain), a peptide targeting MARCKS phosphorylation site domain (PSD) in single and in combination with paclitaxel treatment, on cell proliferation and cell motility in two cell lines of different phenotype (SUM149 for IBC and MDA-MB-231 for nIBC), as well as its distinct molecular mechanisms of action. We also assessed the clinical relevance of the protein expression of MARCKS and phosphatase and tensin homolog (PTEN) in a large series of IBC vs. nIBC patients. Results. In vitro, the treatment with MPS peptide impaired cell proliferation, migration, and invasion in SUM149 compared to MDA-MB-231 cells. More important, MARCKS inhibition increased paclitaxel sensitivity when using combination therapy in SUM149 cells compared to MDA-MB-231 cells. Interestingly, we could partially explain this specific inhibitory effect in IBC cells using western blot: MARCKS inhibition in single and in combination induced up and downregulation of the PTEN/AKT signaling pathway respectively in IBC compared to nIBC cells. Importantly, a negative correlation of MARCKS and PTEN was only found in the clinical IBC samples (180 patients) compared to nIBC samples (355 patients). More importantly, the group of patients with negative MARCKS and positive PTEN protein expression was associated to better 5-year MFS only in IBC patients. Conclusion. These results indicate two major findings: first, the important prognostic value of the negative correlation of MARCKS and PTEN expression in IBC patients, and second the specific role of MARCKS in regulating different downstream pathways and increasing the paclitaxel response in combination treatment in IBC compared to non-IBC. They suggest a potential IBC-specific targetable biomarker, the inhibition of which might impair disease aggressiveness and perhaps enhance patients’ survival. Citation Format: Maroua Manai, Ines Bini, Pascal Finetti, Haifa Bichiou, Carolina Reduzzi, Naziha Ben Hamida, Marc Lopez, Khaled Rahal, Karima Mrad, Mohamed Manai, Massimo Cristofanilli, Hamouda Boussen, Raoudha Doghri, Maher Kharrat, François Bertucci. Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-15.

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.

Towards integration of time-resolved confocal microscopy of a 3D in vitro microfluidic platform with a hybrid multiscale model of tumor angiogenesis

The goal of this study is to calibrate a multiscale model of tumor angiogenesis with time-resolved data to allow for systematic testing of mathematical predictions of vascular sprouting. The multi-scale model consists of an agent-based description of tumor and endothelial cell dynamics coupled to a continuum model of vascular endothelial growth factor concentration. First, we calibrate ordinary differential equation models to time-resolved protein concentration data to estimate the rates of secretion and consumption of vascular endothelial growth factor by endothelial and tumor cells, respectively. These parameters are then input into the multiscale tumor angiogenesis model, and the remaining model parameters are then calibrated to time resolved confocal microscopy images obtained within a 3D vascularized microfluidic platform. The microfluidic platform mimics a functional blood vessel with a surrounding collagen matrix seeded with inflammatory breast cancer cells, which induce tumor angiogenesis. Once the multi-scale model is fully parameterized, we forecast the spatiotemporal distribution of vascular sprouts at future time points and directly compare the predictions to experimentally measured data. We assess the ability of our model to globally recapitulate angiogenic vasculature density, resulting in an average relative calibration error of 17.7% ± 6.3% and an average prediction error of 20.2% ± 4% and 21.7% ± 3.6% using one and four calibrated parameters, respectively. We then assess the model's ability to predict local vessel morphology (individualized vessel structure as opposed to global vascular density), initialized with the first time point and calibrated with two intermediate time points. In this study, we have rigorously calibrated a mechanism-based, multiscale, mathematical model of angiogenic sprouting to multimodal experimental data to make specific, testable predictions.

Synchrotron Fourier-Transform Infrared Microspectroscopy: Characterization of in vitro polarized tumor-associated macrophages stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells

Studies suggested that the pathogenesis of inflammatory breast cancer (IBC) is related to inflammatory manifestations accompanied by specific cellular and molecular mechanisms in the IBC tumor microenvironment (TME). IBC is characterized by significantly higher infiltration of tumor-associated macrophages (TAMs) that contribute to its metastatic process via secreting many cytokines such as TNF, IL-6, IL-8, and IL-10 that enhance invasion and angiogenesis. Thus, there is a need to first understand how IBC-TME modulates the polarization of TAMs to better understand the role of TAMs in IBC. Herein, we used gene expression signature and Synchrotron Fourier-Transform Infrared Microspectroscopy (SR-μFTIR) to study the molecular and biochemical changes, respectively of in vitro polarized TAMs stimulated by the secretome of IBC and non-IBC cells. The gene expression signature showed significant differences in the macrophage's polarization-related genes between stimulated TAMs. FTIR spectra showed absorption bands in the region of 1700–1500 cm−1 attributed to the amide I ν(C=O), & νAS (CN), δ (NH), and amide II ν(CN), δ (NH) proteins bands. Moreover, three peaks of different intensities and areas were detected in the lipid region of the νCH2 and νCH3 stretching modes positioned within the 3000–2800 cm−1 range. The PCA analysis for the second derivative spectra of the amide regions discriminates between stimulated IBC and non-IBC TAMs. This study showed that IBC and non-IBC TMEs differentially modulate the polarization of TAMs and SR-μFTIR can determine these biochemical changes which will help to better understand the potential role of TAMs in IBC.