Browsing by Author "Stack, M. Sharon"
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Item Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer-Like Phenotype(Wiley, 2021-11) Bahcecioglu, Gokhan; Yue, Xiaoshan; Howe, Erin; Guldner, Ian; Stack, M. Sharon; Nakshatri, Harikrishna; Zhang, Siyuan; Zorlutuna, Pinar; Surgery, School of MedicineAge is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer‐like phenotype, while promoting motility and invasiveness in MDA‐MB‐231 cells. Decellularized breast matrix from aged mice leads to loss of E‐cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer‐related proteins. The aged matrix upregulates cancer‐related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial‐mesenchymal transition‐related genes. Lysyl oxidase knockdown reverts the aged matrix‐induced changes to the young levels; it relocalizes E‐cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer‐like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention.Item Aggressive breast cancer in western Kenya has early onset, high proliferation, and immune cell infiltration(Biomed Central, 2016-03) Sawe, Rispah T.; Kerper, Maggie; Badve, Sunil S.; Li, Jun; Sandoval-Cooper, Mayra; Xie, Jingmeng; Shi, Zonggao; Patel, Kirtika; Chumba, David; Ofulla, Ayub; Prosperi, Jenifer; Taylor, Katherine; Stack, M. Sharon; Mining, Simeon; Littlepage, Laurie E.; Pathology and Laboratory Medicine, School of MedicineBackground Breast cancer incidence and mortality vary significantly among different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many nations. Differences in disease progression suggest that aggressive breast tumors may harbor a unique molecular signature to promote disease progression. However, few studies have investigated the pathology and clinical markers expressed in breast tissue from regional African patient populations. Methods We collected 68 malignant and 89 non-cancerous samples from Kenyan breast tissue. To characterize the tumors from these patients, we constructed tissue microarrays (TMAs) from these tissues. Sections from these TMAs were stained and analyzed using immunohistochemistry to detect clinical breast cancer markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2) status, Ki67, and immune cell markers. Results Thirty-three percent of the tumors were triple negative (ER-, PR-, HER2-), 59 % were ER+, and almost all tumors analyzed were HER2-. Seven percent of the breast cancer patients were male, and 30 % were <40 years old at diagnosis. Cancer tissue had increased immune cell infiltration with recruitment of CD163+ (M2 macrophage), CD25+ (regulatory T lymphocyte), and CD4+ (T helper) cells compared to non-cancer tissue. Conclusions We identified clinical biomarkers that may assist in identifying therapy strategies for breast cancer patients in western Kenya. Estrogen receptor status in particular should lead initial treatment strategies in these breast cancer patients. Increased CD25 expression suggests a need for additional treatment strategies designed to overcome immune suppression by CD25+ cells in order to promote the antitumor activity of CD8+ cytotoxic T cells.Item Development and evaluation of ActSeq: A targeted next-generation sequencing panel for clinical oncology use(PLOS, 2022-04-21) Shi, Zonggao; Lopez, Jacqueline; Kalliney, William; Sutton, Bobbie; Simpson, Joyce; Maggert, Kevin; Liu, Sheng; Wan, Jun; Stack, M. Sharon; Medical and Molecular Genetics, School of MedicinePurpose: The demand for high-throughput genetic profiling of somatic mutations in cancer tissues is growing. We sought to establish a targeted next generation sequencing (NGS) panel test for clinical oncology practice. Methods: Customized probes were designed to capture exonic regions of 141 genes selected for the panel, which was aimed for the detection of clinically actionable genetic variations in cancer, including KRAS, NRAS, BRAF, ALK, ROS1, KIT and EGFR. The size of entire targeted regions is 0.8 Mb. Library preparation used NEBNext Ultra II FS kit coupled with target enrichment. Paired-end sequencing was run on Illumina NextSeq 500 at a read length of 150 nt. A bioinformatics workflow focusing on single nucleotide variant and short insertions and deletions (SNV/indel) discovery was established using open source, in-house and commercial software tools. Standard reference DNA samples were used in testing the sensitivity and precision and limit of detection in variant calling. Results: The general performance of the panel was observed in pilot runs. Average total reads per sample ranged from 30 million to 48 million, 73% ~82% unique reads. All runs had more than 99% average mapping rate. Mean target coverage ranged from 727x to 879x. Depth of coverage at 50x or more reached 87% of targeted region and 60% of targeted region received 500x or more coverage depth. Using OncoSpan HD827 DNA, which bears 144 variants (SNV/indel) from 80 genes that are within the targeted region on the panel, our somatic variant calling pipeline reached 97% sensitivity and 100% precision respectively, with near 48 million reads. High concordance with orthogonal approaches in variant detection was further verified with 7 cancer cell lines and 45 clinical specimens. Conclusion: We developed a NGS panel with a focus on clinically actionable gene mutations and validated the performance in library construction, sequencing and variant calling. High concordance with reference materials and orthogonal mutation detection was observed.Item Epigenetic Targeting of Adipocytes Inhibits High-Grade Serous Ovarian Cancer Cell Migration and Invasion(American Association for Cancer Research, 2018-08) Tang, Jessica; Pulliam, Nicholas; Özeş, Ali; Buechlein, Aaron; Ding, Ning; Keer, Harold; Rusch, Doug; O’Hagan, Heather; Stack, M. Sharon; Nephew, Kenneth P.; Medical and Molecular Genetics, School of MedicineOvarian cancer (OC) cells frequently metastasize to the omentum and adipocytes play a significant role in ovarian tumor progression. Therapeutic interventions targeting aberrant DNA methylation in ovarian tumors have shown promise in the clinic but the effects of epigenetic therapy on the tumor microenvironment are understudied. Here, we examined the effect of adipocytes on OC cell behavior in culture and impact of targeting DNA methylation in adipocytes on OC metastasis. The presence of adipocytes increased OC cell migration and invasion and proximal and direct co-culture of adipocytes increased OC proliferation alone or after treatment with carboplatin. Treatment of adipocytes with hypomethylating agent guadecitabine decreased migration and invasion of OC cells towards adipocytes. Subcellular protein fractionation of adipocytes treated with guadecitabine revealed decreased DNA methyltransferase 1 (DNMT1) levels even in the presence of DNA synthesis inhibitor, aphidicolin. Methyl-Capture- and RNA-sequencing analysis of guadecitabine-treated adipocytes revealed derepression of tumor suppressor genes and EMT inhibitors. SUSD2, a secreted tumor suppressor downregulated by promoter CpG island methylation in adipocytes, was upregulated after guadecitabine treatment, and recombinant SUSD2 decreased OC cells migration and invasion. Integrated analysis of the methylomic and transcriptomic data identified pathways associated with inhibition of matrix metalloproteases and fatty acid α-oxidation suggesting a possible mechanism of how epigenetic therapy of adipocytes decreases metastasis. In conclusion, the effect of DNMT inhibitor on fully differentiated adipocytes suggests that hypomethylating agents may impact the tumor microenvironment to decrease cancer cell metastasis.Item In vivo tumor growth of high-grade serous ovarian cancer cell lines(Elsevier, 2015-08) Mitra, Anirban; Davis, David A.; Tomar, Sunil; Roy, Lynn; Gurler, Hilal; Xie, Jia; Lantvit, Daniel D.; Cardenas, Horacio; Fang, Fang; Liu, Yueying; Loughran, Elizabeth; Yang, Jing; Stack, M. Sharon; Emerson, Robert E.; Dahl, Karen D. Cowden; Barbolina, Maria; Nephew, Kenneth P.; Matei, Daniela; Burdette, Joanna E.; Department of Medicine, IU School of MedicineOBJECTIVE: Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. METHODS: To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. RESULTS: Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. CONCLUSIONS: Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community.Item Lysophosphatidic acid modulates ovarian cancer multicellular aggregate assembly and metastatic dissemination(Nature Publishing group, 2020-07-02) Klymenko, Yuliya; Bos, Brandi; Campbell, Leigh; Loughran, Elizabeth; Liu, Yueying; Yang, Jing; Kim, Oleg; Stack, M. Sharon; Obstetrics and Gynecology, School of MedicineEpithelial ovarian cancer (EOC) metastasis occurs by exfoliation of cells and multicellular aggregates (MCAs) from the tumor into the peritoneal cavity, adhesion to and retraction of peritoneal mesothelial cells and subsequent anchoring. Elevated levels of lysophosphatidic acid (LPA) have been linked to aberrant cell proliferation, oncogenesis, and metastasis. LPA disrupts junctional integrity and epithelial cohesion in vitro however, the fate of free-floating cells/MCAs and the response of host peritoneal tissues to LPA remain unclear. EOC MCAs displayed significant LPA-induced changes in surface ultrastructure with the loss of cell surface protrusions and poor aggregation, resulting in increased dissemination of small clusters compared to untreated control MCAs. LPA also diminished the adhesive capacity of EOC single cells and MCAs to murine peritoneal explants and impaired MCA survival and mesothelial clearance competence. Peritoneal tissues from healthy mice injected with LPA exhibited enhanced mesothelial surface microvilli. Ultrastructural alterations were associated with restricted peritoneal susceptibility to metastatic colonization by single cells as well as epithelial-type MCAs. The functional consequence is an LPA-induced dissemination of small mesenchymal-type clusters, promoting a miliary mode of peritoneal seeding that complicates surgical removal and is associated with worse prognosis.Item Modeling the effect of ascites-induced compression on ovarian cancer multicellular aggregates(The Company of Biologists, 2018-09-25) Klymenko, Yuliya; Wates, Rebecca B.; Weiss-Bilka, Holly; Lombard, Rachel; Liu, Yueying; Campbell, Leigh; Kim, Oleg; Wagner, Diane; Ravosa, Matthew J.; Stack, M. Sharon; Mechanical and Energy Engineering, School of Engineering and TechnologyEpithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. EOC dissemination is predominantly via direct extension of cells and multicellular aggregates (MCAs) into the peritoneal cavity, which adhere to and induce retraction of peritoneal mesothelium and proliferate in the submesothelial matrix to generate metastatic lesions. Metastasis is facilitated by the accumulation of malignant ascites (500 ml to >2 l), resulting in physical discomfort and abdominal distension, and leading to poor prognosis. Although intraperitoneal fluid pressure is normally subatmospheric, an average intraperitoneal pressure of 30 cmH2O (22.1 mmHg) has been reported in women with EOC. In this study, to enable experimental evaluation of the impact of high intraperitoneal pressure on EOC progression, two new in vitro model systems were developed. Initial experiments evaluated EOC MCAs in pressure vessels connected to an Instron to apply short-term compressive force. A Flexcell Compression Plus system was then used to enable longer-term compression of MCAs in custom-designed hydrogel carriers. Results show changes in the expression of genes related to epithelial-mesenchymal transition as well as altered dispersal of compressed MCAs on collagen gels. These new model systems have utility for future analyses of compression-induced mechanotransduction and the resulting impact on cellular responses related to intraperitoneal metastatic dissemination.This article has an associated First Person interview with the first authors of the paper.Item Neutrophils Resist Ferroptosis and Promote Breast Cancer Metastasis through Aconitate Decarboxylase 1(Elsevier, 2023) Zhao, Yun; Liu, Zhongshun; Liu, Guoqiang; Zhang, Yuting; Liu, Sheng; Gan, Dailin; Chang, Wennan; Peng, Xiaoxia; Sung, Eun Suh; Gilbert, Keegan; Zhu, Yini; Wang, Xuechun; Zeng, Ziyu; Baldwin, Hope; Ren, Guanzhu; Weaver, Jessica; Huron, Anna; Mayberry, Toni; Wang, Qingfei; Wang, Yujue; Diaz-Rubio, Maria Elena; Su, Xiaoyang; Stack, M. Sharon; Zhang, Siyuan; Lu, Xuemin; Sheldon, Ryan D.; Li, Jun; Zhang, Chi; Wan, Jun; Lu, Xin; Medical and Molecular Genetics, School of MedicineMetastasis causes breast cancer-related mortality. Tumor-infiltrating neutrophils (TINs) inflict immunosuppression and promote metastasis. Therapeutic debilitation of TINs may enhance immunotherapy, yet it remains a challenge to identify therapeutic targets highly expressed and functionally essential in TINs but under-expressed in extra-tumoral neutrophils. Here, using single-cell RNA sequencing to compare TINs and circulating neutrophils in murine mammary tumor models, we identified aconitate decarboxylase 1 (Acod1) as the most upregulated metabolic enzyme in mouse TINs and validated high Acod1 expression in human TINs. Activated through the GM-CSF-JAK/STAT5-C/EBPβ pathway, Acod1 produces itaconate, which mediates Nrf2-dependent defense against ferroptosis and upholds the persistence of TINs. Acod1 ablation abates TIN infiltration, constrains metastasis (but not primary tumors), bolsters antitumor T cell immunity, and boosts the efficacy of immune checkpoint blockade. Our findings reveal how TINs escape from ferroptosis through the Acod1-dependent immunometabolism switch and establish Acod1 as a target to offset immunosuppression and improve immunotherapy against metastasis.Item Tumor infiltrating leukocyte density is independent of tumor grade and molecular subtype in aggressive breast cancer of Western Kenya(BMC, 2017) Sawe, Rispah T.; Mining, Simeon K.; Ofulla, Ayub V.; Patel, Kirtika; Guyah, Bernard; Chumba, David; Prosperi, Jenifer R.; Kerper, Maggie; Shi, Zonggao; Sandoval-Cooper, Mayra; Taylor, Katherine; Badve, Sunil S.; Stack, M. Sharon; Littlepage, Laurie E.; Pathology and Laboratory Medicine, School of MedicineBackground Tumors commonly are infiltrated by leukocytes, or tumor infiltrating leukocytes (TILs). It remains unclear, however, if the density and type of individual TILs has a direct or simply correlative role in promoting poor prognosis in breast cancer patients. Breast cancer in Kenyan women is aggressive with presentation at a young age, with advanced grade (grade III), large tumor size (>2.0 cm), and poor prognosis. We previously observed that the tumors were predominantly estrogen receptor positive (ER+) but also included both a high percentage of triple negative tumors and also increased immune cell infiltration within the tumors. We used breast tumor tissues from each patient to make tissue microarrays that were then stained for leukocyte and myeloid markers including CD4, CD8, CD20, CD25, CD68, and CD163 using immunohistochemical techniques. The immune cell infiltration into the cancer tissue included increased numbers of macrophages (CD68+), helper T cells (CD4+), and CD25+ lymphocytes compared to benign tissue. Results This study characterized the grade, molecular subtypes, and proliferation index of these tumors and determined if TIL density was enriched across any of these factors. We analyzed 49 malignant patient tissue samples for this study. The patient population had a mean age of 51.9 years. The tumors analyzed were heterogeneous by grade: grade I (6%), grade II (47%), and grade III (39%). Most patients presented with large tumors (>2.0 cm) (69%). We classified the tumors into molecular subtypes based on clinical marker expression. Based on this analysis, the molecular subtype distribution was heterogeneous with luminal B (41%), basal/triple negative (TN) (37%), luminal A (14%) and HER2 (8%) breast cancer subtypes. While the basal/TN subtype had a much higher proliferative index (Ki-67+) than did the other molecular subtypes, we did not see a significant correlation between TIL density and either subtype or tumor grade. Therefore, TIL density is independent of molecular subtype and grade. Conclusion This study identified a Kenyan patient cohort that develops large, high-grade tumors primarily of the luminal B and basal molecular subtypes. After analyzing the TILs within these tumors, we found that immune cell infiltration of these tumors correlated with increased proliferation but not grade or molecular subtype. Future research is required to determine if the aberrant recruitment of TILs to tumors contributes to cancer progression and response to cancer treatments.Item The Tumor Microenvironment of High Grade Serous Ovarian Cancer(MDPI, 2018-12-26) Stack, M. Sharon; Nephew, Kenneth P.; Burdette, Joanna E.; Mitra, Anirban K.; Cellular and Integrative Physiology, School of MedicineThe Special Issue on high grade serous ovarian cancer (HGSOC) and the contribution of the tumor micro-environment (TME) consisted of reviews contributed by leaders in the ovarian cancer (OC) field. [...].