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    Annexin A2 mediated TNBC metastasis via small extracellular vesicles: A multi-faceted omics-based characterization
    (2024-03-21) Trivedi, Rucha; Ranade, Payal; Vishwanatha, Jamboor K.
    Purpose – Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer poses a high risk of metastasis due to a lack of targeted therapy and reliance on conventional treatment approaches. Among the organs susceptible to metastasis, the lungs are most frequently affected sites, accounting for approximately 60% of cases. Extracellular vesicles (EV) play a crucial role in establishing a pre-metastatic niche (PMN) for tumor cell homing to secondary sites. Identifying the tumor-derived EV-specific cargo that may potentially alter the lung microenvironment could establish an understanding of the molecular targets contributing to lung PMN development and consequently improving the poor TNBC prognosis and diagnosis. TNBC exhibits elevated expression of Annexin A2 (AnxA2), a plasma and endosome membrane-associated protein. The abundant expression of AnxA2 in tumor tissues and EVs derived from TNBC patients is correlated with poor overall survival and poor distant metastasis-free survival. In previous studies, depletion of AnxA2 protein levels in TNBC-derived EVs resulted in an altered PMN establishment, leading to significantly lower metastatic lesions in the lungs and brain in in vivo system. We aim to evaluate the molecular effects mediated by AnxA2 in TNBC-derived EVs and their cargo that aid the PMN formation and identify underlying molecular targets creating a favorable microenvironment by interaction with lung stroma. Methods and Results – To assess the role of AnxA2 in TNBC lung metastasis, we utilized a lung selective metastatic TNBC cell line, MDA MB 4175 (LM2). We used the shRNA-mediated stable knockdown technique to downregulate AnxA2 protein levels in LM2 cells. We then performed expressional analysis as well as its cell surface functionality using a plasmin generation assay. Additionally, we conducted tumor cell functional assays such as proliferation and invasion assays. Decreased AnxA2 protein levels in LM2 cells significantly reduced its plasmin generation, proliferative ability, and invasive potential. Subsequently, we isolated cell derived small EVs using differential ultracentrifugation and characterized them following the MISEV 2018 guidelines. We confirmed the purity, yield, size, presence of EV-enriched proteins (ESCRT, Hsp, CD9, CD81), and absence of negative proteins (Calnexin, GM130). We then subjected the EVs to quantitative proteomic analysis and identified the differentially expressed proteins upon AnxA2 depletion. We further exposed the healthy lung stromal fibroblasts and the local less-aggressive TNBC cells with EVs to assess the differences in local and distant site uptake and cargo transfer mediated by AnxA2. Conclusion – This comprehensive approach will determine the novel EV-associated proteins that act as functional regulators in promoting TNBC metastasis to the lungs. [This work is supported by the NCI R01CA220273 awarded to Dr. Jamboor K. Vishwanatha]
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    (2024-03-21) Chen, Liling; Sankpal, Umesh; Basha, Riyaz
    Background: Medulloblastoma (MB) is the most common malignant brain tumor in children, with a peak incidence between the ages of 5-9 years old. Originating in the cerebellum, it often metastasizes throughout the CNS via the CSF, making it very difficult to treat. Current treatment options are limited, and includes surgical resection followed by radiation and chemotherapeutic agents. However, these agents are associated with numerous toxicities and long-term neurocognitive deficits in survivors. Our laboratory is interested in identifying drug resistance cell markers and combination therapies that target them to help increase efficacy of the chemotherapeutic agents. We have previously demonstrated that combination therapy of chemotherapeutic agents with Tolfenamic acid (TA), decreased the number of viable cancer cells when compared to the chemotherapeutic agents alone. TA is a non-steroidal anti-inflammatory drug, and its anti-cancer activity can be attributed to its ability to downregulate Specificity Protein 1 (Sp1), a transcription factor responsible for the upregulation of the anti-apoptotic protein, Survivin. Numerous cancerous tumors have been known to express high levels of Sp1 and SurvivIn, however these markers have not been well established in MB. Purpose: The aim of the project is to elucidate the association of Survivin expression in MB cells with the likelihood of patient survival and to test combination treatments of chemotherapeutic agents with the potential Survivin inhibitor, TA. The goal is to reduce the effective dosage of the chemotherapeutic agent Cisplatin, thereby reducing their side effect profiles. Methods: A R2 genomics visualization platform was accessed to obtain data regarding patient survival rates and Survivin expression in MB cells. A Kaplan-Meier curve was then generated to analyze the relationship. MB cell lines, DAOY, and D283 cells were obtained through the ATCC and cultured following standard cell culture conditions. Cells were then treated with vehicle (DMSO) or optimized doses of a chemotherapeutic agents (Vincristine or Cisplatin) with TA. Cell viability was measured at 24h and 48h post-treatment using Cell-TiterGlo kit (Promega) following manufacturer’s instructions. Results: The Kaplan-Meier curve showed that the overexpression of Survivin resulted in a poor prognosis and low survival rates among MB patients. Compared to results of MB cell inhibition with individual agents (Vincristine and Cisplatin) from our previous studies, the combination of TA and Vincristine or TA and Cisplatin showed decreased MB cell growth and downregulation of Survivin. Differential effects of Vincristine and Cisplatin were noted against DAOY and D283 cell lines. Conclusion: These preliminary observations suggest that Survivin expression may be associated with poor prognosis in MB patients and that inhibiting Survivin with TA is inducing the anti-proliferative effects of chemotherapeutic agents in MB cells. Further research involving other chemotherapies is required to understand TA’s role in Survivin inhibition. Understanding the specific effect of Cisplatin can help to design the therapies based on the molecular sub-group of MB.
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    Treatment Patterns and Survival Outcomes of Immune Checkpoint Inhibitors in Advanced Non-Small Cell Lung Cancer Survival at a Safety-Net Hospital
    (2024-03-21) Sabloak, Krishti; Derton, Abigail; Teigan, Kari; Gonzalez, Mario; Ghabach, Bassam; Narra, Kalyani
    Background: Immune checkpoint inhibitors (ICI) have transformed the treatment of advanced non-small cell lung cancer (aNSCLC) without driver mutations. KEYNOTE 189 trial showed median overall survival (OS) of 21.8 months (m) for chemotherapy with ICI (CT-ICI) compared to 12.1m for chemotherapy (CT). Since a large percentage of underserved cancer patients in the United States receive care from safety-net hospitals (SNH), this poses the question: Are these patients benefitting from ICI? Herein, we report the treatment and survival patterns of patients with aNSCLC at John Peter Smith Hospital (JPS), a SNH in North Texas. Methods: Patient data were obtained from the JPS tumor registry for this retrospective study. Eligible patients were diagnosed at JPS from 1/1/2017 to 12/31/2021 with stages IIIB/IIIC/IV NSCLC. Patients with driver mutations were excluded. Electronic records were reviewed for programmed death ligand 1 (PD-L1) testing and first-line treatments. OS was calculated from diagnosis to death (if applicable) or the last chart entry before 5/31/2023. Covariates were sex, race, age at diagnosis, stage, histology and PDL-1. Log-rank tests were used to compare survival distributions. OS probability within each treatment group was modeled using a Kaplan-Meier curve. The log-normal accelerated failure time model was used to estimate the effects of covariates on survival. Results: 195 patients were included: 48% Non-Hispanic White, 35% Black, 12% Hispanic, and 6% Asian. 59% were males. 81% of patients had stage IV disease. Treatments were as follows: 29 CT, 27 CT-ICI, 15 ICI, and 15 chemoradiation (CRT). 106 (54%) patients did not receive any treatment (NT). CRT was used in 75% of treated stages IIIB/IIIC. Median OS for CT, CT-ICI, ICI, CRT, and NT were 6.6m, 22.6m, 23.6m, 23.4m, and 2.7m, respectively. The log-rank test detected a statistically significant difference in OS between CT-ICI and CT (p < 0.001) and between ICI and CT (p=0.003) but no difference between CT-ICI and ICI (p=1). 66% of patients underwent PDL-1 testing: PDL-1 <1%, PDL-1 1-49%, and PDL-1 ≥ 50% had longer OS with p=0.02, p=0.01 and p<0.001 respectively compared to those not tested. Conclusions: Our study is the first in SNH population, with OS similar to published trials. aNSCLC patients who received first-line CT-ICI or ICI had better survival compared to CT alone regardless of PDL-1 results. We should study the reasons why most patients did not receive any treatment and extend the benefit of ICIs to as many patients with aNSCLC as possible.
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    Immunotherapy in Pediatrics
    (2024-03-21) Aguilar, Rocio; Wojciechowska, Natalia; Murray, Lauren; Ray, Anish
    Background: Immunotherapy has emerged as a promising treatment approach, specifically designed to boost the response of the immune system to target tumor cells while sparing nontumor tissue. This treatment not only holds the potential for enhanced efficacy but also translates into reduced adverse effects on patients while improving outcomes. Despite its early success, the application of immunotherapy has largely been confined to adults, with slow adoption noted in the treatment of pediatric malignancies. Case Information: Here the objective is to demonstrate a single institution’s experience with immunotherapy in pediatric oncology, to further the exploration of these treatment modalities in the treatment of children with cancer. In our review, we studied a database of 48 pediatric patients out of 226, expressing programmed death ligand 1 (PD-L1), of which 7 have undergone immunotherapy. In this project, we studied various tumor types in which immunotherapy found its utilization. We also noted adverse effects associated with this particular therapy as well as efficacy. We believe immunotherapy is poised to deliver a positive impact in the realm of pediatric patients. 7 pediatric patients with positive expression of PD-L1 received immunotherapy, involving nivolumab alone or in combination with ipilimumab or brentuximab. Hodgkin lymphoma (n=2), metastatic melanoma (n=2), histiocytic sarcoma (n=1), rectal carcinoma in the context of constitutional mismatch repair deficiency (cMMRD) (n=1), epithelioid and spindle cell hemangioma (n=1) comprised the diagnoses. Patients received between 4 and 18 cycles of immunotherapy. Out of all patients who had completed their immunotherapy regimens (n=5) or remained on treatment (n=2), 6 achieved remission or had stabilized disease. The diagnoses responding to treatment included Hodgkin lymphoma (n=2), metastatic melanoma (n=2), rectal carcinoma due to cMMRD (n=1), and epithelioid and spindle cell hemangioma (n=1). Unfortunately, after 13 months in remission, the patient suffering from histiocytic sarcoma experienced reoccurrence in the duodenum. Immune-related adverse events included mild allergic reactions, prodromal symptoms, anemia, neutropenia, transaminitis, endocrinopathies, and self-limiting neuritis. Conclusions: This report highlights the positive impact immunotherapy can have in the realm of pediatric malignancies, with the possibility of tumor regression or stabilizing disease. Further research is needed to accurately identify pediatric oncology patients that could benefit from immunotherapy.
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    Targeting Sp1 in Ewing Sarcoma: A multi-approach method for the utilization of Mithramycin
    (2024-03-21) Lambring, Christoffer; Sankpal, Umesh; Behera, Santosh; Basha, Riyaz
    Purpose: Ewing Sarcoma (ES) is a bone and soft tissue cancer affecting young adults and children. ES mostly occurs in the bones or soft tissue of the arms, legs, and pelvis. Localized ES presents with 5-year survival rate of 70%, but metastatic 5-year survival rate is between 15% and 30%. Our laboratory is interested in combination treatments using less toxic agents to induce sensitization to chemotherapy in ES. The anti-cancer activity of an antineoplastic antibiotic, Mithramycin, against ES cells has been shown. Mithramycin inhibits Specificity protein 1 (Sp1) a marker associated with aggressive cancer cell growth and resistance to chemo/radiation therapies. However, its mechanistic effects on other oncogenic proteins have yet to be elucidated in ES. The purpose of this study is to evaluate the effectiveness of Mithramycin and various combinations with other chemotherapeutic agents, Etoposide and Vincristine, to inhibit ES cell growth and assess the effect on key cancer related proteins regulated by Sp1. Future studies will include expanding upon Mithramycin’s mechanism of action in Ewing Sarcoma utilizing proteomics and various computational methods. Methods: Cell lines were obtained from Children’s Oncology Group (COG). Anti-proliferative activity of Mithramycin and/or Vincristine and Etoposide against ES cell lines, TC205 and CHLA10, was evaluated using CellTiterGlo kit. Dose curves were plotted and IC50 values were determined by Sigma-Plot software. The expression of Sp1 was determined by Western blot analysis. The specific type of effect (additive/antagonistic/ synergistic) of the combination treatments were determined by analyzing the combination index obtained via Calcusyn software. Nude mice were injected with TC205 cells and treated over two weeks with either Mithramycin (1mg/kg per week) and/or Etoposide (5mg/kg per week) and tumor volume was compared. Protein models were obtained from UnitProtKB and PDB and molecular dynamics simulations were run in the Schrödinger platform. Results: Mithramycin, etoposide, and vincristine decreased ES cell line viability in TC205 and CHLA10 cells as monotherapies, but more effectively in combination. Tumor volume was greatly attenuated upon Mithramycin and/or etoposide introduction, but more significantly when used in combination. Decreases in viability upon chemotherapeutic and Mithramycin introduction were drastically increased when used in combination and this effect was mirrored in further decreases in Sp1 expression. Synergistic drug responses were shown in the combination of Mithramycin with both Vincristine and Etoposide (CI <1). Sp1, Sp3, and survivin protein models were established and binding scoring and identification of key residues in Mithramycin protein interactions were identified. Conclusions: Mithramycin may effectively sensitize ES cells and improve the response of chemotherapy while lowering necessary effective dosages. Studies to understand the mechanism of action of Mithramycin on Sp1, Sp3, survivin, and other proteins involved in Ewing Sarcoma are underway.
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    Elucidating the role of MIEN1 in colorectal cancer metastasis
    (2024-03-21) Ranade, Payal; Trivedi, Rucha
    Purpose: Colorectal cancer (CRC) ranks among the foremost contributors to global cancer-related fatalities, with its progression marked by hyperproliferation of epithelial cells culminating in polyp formation. Stages 3 and 4 of CRC are notably associated with metastasis. Despite existing chemotherapy focusing on symptom attenuation, a definitive cure remains elusive. This study addresses the imperative need to comprehend the underlying mechanisms of CRC progression for timely diagnosis and effective treatment. The process of metastasis involves a complex interplay of signaling pathways, where various proteins play crucial roles. Migration and Invasion ENhancer 1 (MIEN1) is a significant protein facilitating migration, and its expression is notably upregulated in CRC tissue compared to normal colorectal tissue, correlating with invasive behavior. The exact mechanism of MIEN1 involvement in metastasis remains unexplored. MIEN1 overexpression results from 17q12 chromosomal amplification, and dysregulated expression is associated with trans-regulation of its minimal promoter region. This study aims to investigate the impact of MIEN1 promoter ablation on CRC migration properties. Methods: Utilizing CRISPR-Cas9 gene editing, we deleted the MIEN1 promoter region in the CRC cell line HT29. RNA sequencing and bioinformatics tools revealed differentially expressed genes (DEGs) associated with essential biological processes and molecular pathways, including cell adhesion, migration, invasion, and angiogenesis. Evaluation of critical genes in CRC biogenesis at RNA and protein levels demonstrated that MIEN1 knock-out significantly reduced the migration potential of HT29 cells, as evidenced by functional assays such as wound healing and Matrigel invasion. Furthermore, MIEN1 deletion disrupted cytoskeletal rearrangement, affecting F-actin reorganization, confirmed through phalloidin staining. Confocal staining of proteins involved in actin cytoskeleton rearrangement provided insights into MIEN1's role in mediating phosphorylation of FAK at different sites. Immunoblotting analysis further substantiated MIEN1's involvement in actin cytoskeleton dynamics. Results: Our findings indicate a significant reduction in the migration potential of HT29 cells in the absence of MIEN1 protein. Additionally, we demonstrated the disruption of cytoskeletal rearrangement, specifically affecting F-actin reorganization, upon MIEN1 deletion using phalloidin staining. Confocal staining of various proteins involved in actin cytoskeleton rearrangement, such as FAK and cofilin, further strengthened the evidence of MIEN1 in actin reorganization. Immunoblotting analysis of a diverse set of proteins further confirmed the involvement of MIEN1 in the dynamics of actin cytoskeleton. Conclusion: MIEN1, an influential metastatic protein overexpressed in cancerous cells, plays a crucial role in various signaling pathways governing CRC migration. Deletion of MIEN1 perturbs biological processes, particularly actin cytoskeleton rearrangement crucial for metastasis. Thus, targeting MIEN1 emerges as a promising therapeutic strategy for CRC patients.
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    Is there a relationship between Religious Affiliation and Adherence to Recommended Cervical Cancer Screening Guidelines?
    (2024-03-21) Krenek, Brittany; Akpan, Idara; Balasundaram, Rohit; Thompson, Erika; Luningham, Justin
    Purpose: Cervical cancer is a preventable disease that, despite growing research and health advancements, remains a public health concern in the United States (US).The United States Preventative Services Task Force (USPSTF) recommends cervical cancer screenings (CCS) for individuals with a cervix aged 21 to 65 years of age. Adherence Guidelines recommend cytology tests (Pap smears) for individuals 21 to 29 years of age and cytology tests alone or with a high-risk human papillomavirus (hrHPV) test for individuals aged 30 to 65 years old. In 2023, 74.2% of US-eligible adults were determined to adhere to these guidelines. Research has indicated many influential social and cultural factors of CCS adherence, including religion. However, to our knowledge, no studies have been conducted in the US to assess the specific influences of Christian denominations on CCS adherence. Research indicates that discussions about sexual health may be stigmatized among Christian denominations in the US. Additionally, testing for HPV may be viewed as a rejection of abstinence, which is advocated by my many Christian denominations, further applying social stigmas to CCS screening. Therefore, this study explored associations between CCS guideline adherence and religious denomination affiliation, accounting for other demographic characteristics. Method: Women aged21-49 years old (n =4561) were examined for USPSTF CCS adherence from the National Survey of Family Growth (2017-2019). A weighted multivariable logistic regression was used to examine the likelihood of adhering to CCS guidelines by Christian denomination compared to no religious affiliation, controlling for race/ethnicity, education, and age group. Adherence was determined by the last reported Pap test and last reported HPV test, with up-to-date status varying across age groups in accordance with USPSTF guidelines (21-29 years old compared to 30+ years old). Women reporting a history of a hysterectomy were excluded from the analysis. Results: Overall, 56.3% of participants adhered to the CCS guidelines. The rates of adherence to CCS guidelines across the available Christian denominations were 65.8% among Black Protestants, 59.0%amongMainline Protestants, 53.2% among Catholics, 49.6% among Evangelical Protestants,49.3%for those of another Religion, and 58.7% for those with no religious affiliation. Evangelical Protestants and individuals self-identifying with non-Christian religions had lower odds of screening guidelines compared to those with no religious affiliation(OR = 0.80, 95%CI [0.66, 0.98]and OR=0.67,95%CI [0.52,0.86],respectively).Women who self-identified as non-Hispanic Black were more likely to adhere to CCS guidelines than non-Hispanic White women(OR= 1.96, CI [1.52, 2.53]).Women 30 years or older were less likely to adhere than 21–29-year-olds (OR = 0.19,CI [0.16,0.22]).Compared to individuals who obtained bachelor's degrees, there was no significant association with the odds of CCS adherence among other educational levels in the multivariable model. Conclusion: Associations were observed between adhering to CCS guidelines and religious denomination after accounting for race/ethnicity, age, and education. Women who were Evangelic Protestants or part of a non-Christian religion were less likely to adhere. Additional studies should further evaluate associations and advise culturally tailored campaigns to reduce CCS stigmas and increase overall adherence.
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    Osteoblastoma: A Case Series
    (2024-03-21) Murray, Lauren; Aguilar, Rocio; Ray, Anish; Wojciechowska, Natalia
    Background: Osteoblastoma (OB) is a rare tumor constituting only 1% of all primary bone tumors. It typically occurs in males, in the second decade of life, and primarily affects the vertebral column and long bones. While it is generally considered benign due to its slow-growing nature and inability to metastasize, OB can occasionally exhibit aggressive features of growth, causing significant bone destruction, infiltration of the soft tissues, and epidural extension. The current treatment of choice is complete surgical resection, reserving radiotherapy, and chemotherapy for aggressive or surgically unresectable tumors. Next-generation sequencing (NGS) is not included in the typical evaluation and may offer novel insight into genetic alterations associated with OB. Case Information: With the description of these three cases, we aim to highlight the extraordinarily diverse ways that OB can present and behave, from a highly aggressive course in the cervical spine that progressed even after 4 surgeries to a dormant disease affecting the clavicle, requiring only local curettage for diagnosis and no further treatment. Additionally, we present genetic information acquired by NGS testing, not conventionally part of the work-up for OB. This case series examined NGS results, surgical margin outcomes, treatment modalities, imaging, and relevant scientific literature for each case, as well as pertinent information from each patient’s medical history. Of the three cases we reviewed, all were female, ages 8, 9 and 11. Tumor locations varied, affecting the thoracic vertebrae T8-9, cervical vertebra C7, and clavicle. The disease courses of these patients ranged from inoperable spinal tumor necessitating radiotherapy to residual disease after 4 vertebrectomy surgeries to dormant disease of the clavicle with resolution after curettage. Furthermore, the differential diagnoses considered among these cases consisted of primary aneurysmal bone cyst, chondroblastoma, osteosarcoma, Ewing sarcoma, lymphoma, rhabdomyosarcoma, giant cell tumor, and chronic recurrent multifocal osteomyelitis, demonstrating the potential difficulty in diagnosing OB. NGS testing was performed on tumor samples of 2 out of 3 of the patients and revealed a WWTR1-FOSB translocation and a germline ATM mutation. The two patients with vertebral involvement experienced progressive disease within 7 months postoperatively and the remaining patient presented with normally maturing bone 3 months postoperatively. Conclusions: This case series emphasizes the capability of OB to present and progress in incredibly diverse ways. Moreover, there is currently a lack of knowledge regarding the genetic drivers of this disease process and we believe that NGS testing may assist in furthering the genetic characterization of this tumor.
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    Appendiceal Mucinous Neoplasm Case Study
    (2024-03-21) Tamayo, Jesús; Patterson, Tyler
    Appendiceal Mucinous Neoplasm Case Study Jesse Tamayo, Tyler Patterson, Michael Smith, Jay Patel Appendiceal Mucinous Neoplasms account for less than 1% of all malignancies in the United States. Patients with low grade neoplasms often present with symptoms of an acute appendicitis with right lower quadrant pain due to the distention of the appendix caused by the increasing levels of mucin. These patients may also have perforation of the appendix or appendicitis if the tumor growth is obstructing the orifice of the appendix. An patient with advanced disease may present with similar symptoms as well as distention of the abdomen due to mucinous ascites growing in the peritoneum. Patients may also present with weight loss, abdominal pain, and even intestinal obstruction. A majority of these primary neoplasms have mucin involved in more than 50% of the lesion, arising from low-grade appendiceal neoplasms (LAMN) seen as adenomatous changes in appendiceal mucosa. They can also arise from a polyp forming adenoma similar to those seen in patients who develop colon cancer. Histologically they can be seen with signet ring cells. The patient involved in this case report had presented to the Emergency Department on numerous occasions for right lower quadrant pain. This pain had persisted for years, CT imaging was performed and the tumor was recognized. A procedure for removal was scheduled, however prior to the day of surgery, the patient again presented to the emergency department with excruciating RLQ pain. Surgery was performed the following day. The tumor size was so great that the patient also had a right hemicolectomy performed to remove the tumor in entirety. It was 5.5 cm in greatest dimension. Surgical margins were negative for invasion and twenty six lymph nodes were biopsied and all came negative for cancer. The surgical case was successful for a appendectomy along with a right hemicolectomy to remove a 5.5 by 1.5 low grade appendiceal mucinous tumor confined to the muscularis propria of this patient.
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    LN-229 glioblastoma multiforme cell line expresses CD155 (PVR), a target for Natural Killer Cell-mediated immunotherapy
    (2024-03-21) Cooksey, Luke; Mathew, Porunelloor; Mangan, Enrique
    Purpose: Glioblastoma multiforme (GBM) is the most common form of primary brain cancer in adults. Currently, it has a dismal five-year survival rate of less than 7%. Current commonplace treatment options include surgery, chemotherapy, and radiation. In recent years, there has been a major attempt at bringing the immunotherapy revolution into the treatment pillars for GBM. Immunotherapies depend upon targeting molecular antigens that are unique to cancer cells. Our lab is currently working to identify novel antigens on GBM as future candidates for Natural Killer (NK) cell-mediated immunotherapy. In several cancers, the surface antigen known as CD155 (PVR) has been shown to be overexpressed and is important as part of a mechanism for cancer cells to evade NK cell and T cell-mediated immune responses. Typically, CD155 is not highly expressed on healthy cells, making it an attractive immunotherapy target. We set out here to investigate the expression of CD155 on LN-229 cells. Methods: We examined the expression of CD155 on the LN-229 GBM cell line using flow cytometry utilizing PE-labeled antibodies specific for CD155. Our hypothesis: LN-229 cells would show increased detection of fluorescence signal (from anti-CD155 antibodies) when compared to the fluorescence detection of negative control groups (no staining group and PE-isotype control group). Results: CD155 detected on LN-229 cells via the detection of increased fluorescence signal (versus negative controls). Conclusions: Based on our results, we concluded that CD155 is overexpressed on the LN-229 cell line and is a sufficient candidate for studying NK cell-mediated immunotherapy in in vitro contexts using LN-229 cells. Currently, we are evaluating blocking inhibitory signals to NK cells mediated through the CD155-TIGIT interaction to target GBM for NK cytotoxicity in LN-229 cells.
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    Survivin as a Prognostic Marker in Breast Cancer
    (2024-03-21) Khosla, Aishani; Sharna, Ansley; Sankpal, Umesh
    Purpose: Breast cancer is the second leading cause of cancer-related death for women in the United States and continues to pose a threat to millions of women globally. Incidence rates of breast cancer continue to rise across ethnic/ racial groups and survival rates continue to increase with the development of screening protocols and targeted therapeutics. However, despite the advances in disease diagnosis and management a racial disparity continues to be evident. Even with advanced therapeutics and the development of various prognostic markers, Black women continue to have higher rates of breast cancer mortality when compared to other racial groups. Mortality is also correlated with the status of metastasis, as advanced disease points to poor outcomes and survival rates. The evident racial disparity and advanced staging reflect the need for new and more efficient prognostic markers to be developed to better manage and treat breast cancer. Existing prognostic factors include immunohistochemical markers such as estrogen receptor, progesterone receptor, Ki-67, and human epidermal growth factor receptor-2. One potential immunohistochemical marker is the Inhibitor of Apoptosis Protein (IAP), survivin, with elevated levels of expression found in various cancer types, including breast cancer. Previous research has linked elevated levels of survivin expression with increased resistance to therapeutics like chemotherapy and radiation. However, there remains some question of the true clinical significance of survivin as a prognostic factor. The objective of this literature review is to provide an overview of research on the use of survivin expression as a clinically useful prognostic marker in breast cancer. Methods: Relevant research articles were collected mainly through the PubMed database. Studies investigating survivin expression alone or with other prognostic markers of breast cancer using immunohistochemistry or PCR analysis of breast tumor tissue were selected. Results: Review of this literature showed that the majority of studies found a significant correlation between levels of survivin expression and poor prognosis. Fourteen studies from the total seventeen analyzed identified various established prognostic markers to be correlated with survivin expression. Increased levels of survivin were associated with poor prognosis through findings of higher grade, greater lymph node metastasis, increased proliferation, and other indicators. Conclusion: These findings suggest that survivin has the potential to be used clinically in combination with other biomarkers to bolster diagnostics. In addition, the results indicate that survivin may be used as a marker in disease management as well as a unique focus of targeted therapy.
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    Increasing Cancer Genetic Referrals via Best Practice Alerts (BPA)
    (2024-03-21) Reddy, Priyanka
    Background: Germline genetic testing (GT) is recommended for all patients (pts) with pancreatic cancer (PC). However, genetic referral (GR) rates for PC are low even at academic centers; there is a paucity of literature at safety-net hospitals (SNH) which often treat higher percentages of cancers at advanced stages. Providers focus on the challenging treatment of PC and may not prioritize GT referrals. Technology can be adapted to aid in this process. Best practice alerts (BPA) built in Electronic Medical Records (EMR) can prompt providers to place GR order. We initiated BPA at John Peter Smith (JPS) hospital, a SNH in Texas, and evaluated if the BPA improved referral patterns. Methods: A retrospective review was conducted using JPS tumor registry data. We studied pts diagnosed with pancreatic adenocarcinoma (AC) or neuroendocrine tumors (NET) from 1/1/2018 to 4/30/2023 at JPS. BPA go-live date was 10/19/2022. Data Analytics team queried EMR regarding GR, if pts completed their genetics encounter (GE), and GT. Statistical analysis using a two-tailed T test was performed. Results: 162 PC pts were identified: 155 (96%) AC and 7 (4%) NET, and the median age was 59 years. A majority of patients were diagnosed in house (114/162, 70%), while the rest were referred to JPS after diagnosis elsewhere. A total of 57 pts were excluded due to being placed on hospice, death within 3 months of diagnosis, or refusing oncology services at JPS. 105 pts were included for analyses: 96 were diagnosed with PC before the BPA go-live date (pre-BPA) and 9 after the BPA go-live date (post-BPA). 36 (38%) pts were referred pre-BPA. 23 pre-BPA pts completed GE, median of 15 days from referral. 7 out of 9 (78%) pts received GR post-BPA. Post-BPA pts were all referred due to BPA triggers being fired. 2 post-BPA pts completed GE with a median time of 27.5 days from GR. Since launching the BPA, a statistically significant increase in GR has been observed (p=0.0187). 2 pre-BPA pts who completed GT were found to have a pathogenic variant. Conclusions: BPA has shown a significant improvement in GR for PC. At the recent 2024 quarterly JPS Cancer Committee meeting, we found an update of considerable improvement in genetic referrals, with 83% of new pancreatic cancers receiving genetic referrals in 2023 compared to only 35% in 2022.Our study population is limited by rarity of PC, and the BPA alert is set to only fire for pts receiving oncology care at JPS. We noted a large number of pre-BPA pts that have yet to be referred and remain at risk. We plan to have a process to notify these pts’ providers to refer them to genetic testing. For future studies, we will analyze pts with other cancer diagnoses to continue to improve upon the GR process. Authors: Priyanka Reddy, Jacqueline Mersch, Melissa Howell, Jolonda Bullock, Brian Reys, Parker Read, Nitin Rajpurohit, Bassam Ghabach, Kalyani Narra
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    Assessing Cancer Health Disparities Within Racial/Ethnic Groups in Tarrant, Dallas, and Bexar County
    (2024-03-21) Jayesh, Irene; Chu, Megan; Nava, Marcela
    Background: Cancer health disparities refers to significant differences in cancer incidence, prevalence, death, survivorship, and burden that exist among different population groups in the United States. Those who have limited or lack access to effective health care are often impacted more severely by cancer compared to the general population. Social and economic factors such as insurance status, income level, neighborhood poverty, and education level also play a large influence in cancer disparities observed among groups. Strong public health policies can serve an important role in reducing cancer health disparities by tackling prevention and early detection, especially within vulnerable populations. Health disparities are particularly pronounced in racial/ethnic groups such as African Americans and Latinos, and even persist when comparing groups of similar socioeconomic status. The findings of this research project will provide valuable information regarding the urgent need to address cancer disparities within Texas counties, especially Tarrant County, Dallas County, and Bexar County. Purpose: Highlight disparities in the frequency and rate of cervical, breast, prostate, lung, and colorectal cancer among racial/ethnic groups within Tarrant, Dallas, and Bexar county to increase awareness for policy making. Methods: County-level cancer data was pulled from the Surveillance, Epidemiology, and End Results (SEER) cancer registry. The statistics in this registry were categorized by the patient's racial group. Focusing only on statistically significant changes, we ranked all counties by their average annual percent change (APC) from 2000-2020 trend for frequency and age-adjusted rate per 100,000 population for cervical, breast, prostate, colorectal, and lung cancer. Results: In Tarrant County, the annual percent change of cancer frequency between 2000-2020 were as follows: 2.5% increase for cervix, 2.7% increase for breast, 0.9% increase for prostate, 1.3% increase for colorectal, and 0.6% increase for lung cancer. Also in Tarrant County, the annual percent change of cancer rates from 2000-2020 were as follows: 0.2% increase for cervix, 0.2% increase for breast, -3.1% decrease for prostate, -2.2% decrease for colorectal, and -2.8% decrease for lung cancer. Tarrant county was also ranked 1st in Texas and 5th in the nation for cervical cancer in Non-hispanic black. Dallas county was ranked 7th in Texas and 15th in the nation for cervical cancer in Hispanics. Conclusion: This data brings awareness to certain groups within Tarrant County, Dallas County, and Bexar county that are more likely to be diagnosed with cancer mainly because of their ethnicity. These findings bring up crucial discussions about the underlying factors contributing to the increase in cancer risk among specific ethnic populations, whether it is a genetic association or a socioeconomic association. For example, despite initiatives taken to tackle this disparity, the data shows that Hispanic women are still highly at risk for developing cervical cancer across all three counties. Policymakers and advocates can use this data to identify which type of cancers and ethnic groups require focused intervention and attention in the community.
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    Expression of CD155 on LN-18 Glioblastoma Multiforme Cancer Cell Line
    (2024-03-21) Mangan, Enrique
    Purpose: Glioblastoma multiforme (GBM) stands as the most common form of primary brain cancer among adults, it has a five-year survival rate of 5% - 10%. Traditional treatment options include surgical intervention, chemotherapy, and radiation therapy. Recently, immunotherapy has emerged as a promising treatment paradigm for cancer. These immunotherapies often rely on targeting molecular antigens unique to cancer cells. Surface antigens such as CD155 (PVR), previously thought to be expressed almost exclusively by stem cells, can also be expressed on the surface of certain cancer cells. CD-155, in the context of cancer survival, acts to downregulate cancer killing mechanisms of Natural Killer (NK) cells, making it a promising target for immunotherapy. To treat GBM tumors lacking expression of established antigens, our laboratory has embarked on the quest to uncover novel antigens specific to GBM, envisioned as candidates for Natural Killer (NK) cell-mediated immunotherapy. Methods: This experiment investigated the potential expression of cell surface CD155 on the LN-18 glioblastoma cell line utilizing flow cytometry, employing PE-labeled antibodies designed for CD155 detection. To ensure accuracy, we employed two negative control groups for this cell line: 1) unstained LN-18 cells and 2) LN-18 cells stained with PE-mIgG2a isotype control antibodies. Our hypothesis posited that LN-18 cells would exhibit a heightened fluorescence signal from anti-CD155 antibodies compared to the fluorescence levels observed in the negative control groups (unstained and isotype control-stained cells). Results and Conclusions: Based on the detection of increased fluorescence signal versus negative controls, CD155 was identified to be expressed on LN-18 cells.
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    Exploring Racial/Ethnic Disparities in Multimorbidity Among Cancer Patients at a Safety-Net Health System
    (2024-03-21) Kalman, Kyra; Singh, Anand; Meadows, Rachel
    Purpose: Multimorbidity, defined as having two or more chronic health conditions, can substantially impact quality of life and mortality. Multimorbidity also increases the complexity of healthcare and treatment decision-making, particularly during cancer treatment. Racial/ethnic minorities may have higher risk for multimorbidity, potentially attributed to socioeconomic or other health-related factors. Therefore, we aimed to investigate racial/ethnic disparities in multimorbidity and potential explanatory factors among newly diagnosed cancer patients at a safety-net health system. Methods: We used electronic health record and cancer registry data from JPS Health Network, an urban safety-net health system in North Texas. Our eligible population included patients ≥18 years of age diagnosed with a first primary cancer in 2016–2020 (excluding in situ cases), whose initial diagnosis or at least part of the first course cancer treatment was received at JPS. Our outcomes were prevalence of multimorbidity (≥2 chronic conditions) and severe multimorbidity (≥3 chronic conditions). We included 30 chronic conditions defined by the Centers for Medicare & Medicaid Services and used up to a 2-year lookback period before cancer diagnosis. We used descriptive statistics to summarize sociodemographic characteristics and assess the proportions of multimorbidity by race/ethnicity. We used logistic regression to assess unadjusted and adjusted associations between race/ethnicity and multimorbidity and severe multimorbidity. Results: Our study included5,019 patients newly diagnosed with cancer. The most common cancer types were breast (13%), lung (12%), and colorectal (11%). The median age was 58 years (interquartile range: 50 – 64) and the majority were females (51%), racial/ethnic minority (59%), and uninsured (51%).Overall, 79% had multimorbidity and 62% had severe multimorbidity. Non-Hispanic Black (NHB) patients had the highest proportion of multimorbidity (86%) and severe multimorbidity (73%),whereas Hispanic patients had the lowest proportions (74% and 54%, respectively). In our unadjusted logistic regression model for multimorbidity, NHB patients had 1.75[95% CI: 1.44, 2.12]times higher odds and Hispanic patients had 0.80[95% CI: 0.69, 0.94]times the odds of multimorbidity compared with non-Hispanic White (NHW) patients. After adjusting for age, sex, body mass index, insurance status, and marital status, NHB patients maintained higher odds of multimorbidity (1.77 times [95% CI: 1.45, 2.17]), but Hispanic patients had no substantial difference (OR: 0.91[95% CI: 0.79, 1.07]) compared with NHW patients. In the unadjusted model for severe multimorbidity, NHB patients had 1.68 [95% CI: 1.44, 1.96]times higher odds and Hispanic patients had 0.71 [95% CI: 0.62, 0.81] times the odds of severe multimorbidity compared with NHW patients. In our adjusted model, NHB patientshad1.66 [95% CI: 1.41, 1.96] times higher odds and Hispanic patients had 0.77 [95% CI: 0.66, 0.89]times the odds of severe multimorbidity compared with NHW patients. Conclusions: We observed substantial racial/ethnic disparities in multimorbidity and severe multimorbidity among newly diagnosed cancer patients at a safety-net health system. Racial/ethnic disparities did not decrease after controlling for sociodemographic factors. Additional factors that are currently undocumented in electronic health records (e.g., social determinants of health) may help explain racial/ethnic disparities in multimorbidity and identify targetable points for interventions.
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    GW525701 Treatment Alters Cellular Morphology and Decreases Triple-Negative Breast Cancer Viability
    (2024-03-21) Rinderle, Caroline; Bunnell, Bruce
    Purpose: Breast cancer is the second leading cause of death in women in the United States. Triple-negative breast cancer (TNBC) is named for its lack of estrogen (ER) and progesterone (PR) receptors, as well as HER2. The lack of receptors makes these tumors increasingly more difficult to treat, resulting in worser clinical outcomes compared to other receptor-positive subtypes. Kinase inhibitor compounds have been of great interest for treating TNBC patients. Specific kinase inhibitors have been successful against other cancer subtypes, namely chronic myeloid leukemia (CML), which has substantially bettered the outcomes associated with this disease. However, in total, only 72 kinase inhibitors have been approved for clinical use, many of which are not used as cancer therapeutics at all, let alone for breast cancer. Much more research remains to determine the role of other, understudied kinases in cancer proliferation and survival. One kinase inhibitor compound, GW525701, targets STK10, TNIK, SLK, MAP4K4, MINK, and DDR1, none of which are targeted by FDA-approved therapeutics. If treating cancer cells with this kinase inhibitor compound reduces cancer survival and proliferation, then it may be a potential future therapeutic, and will give better insight into the mechanisms controlling TNBC survival. Methods: Breast cancer cells (4IC, BT-549, MCF-7) were treated with GW525701 for 72 hours at a range of concentrations (1nM, 10nM, 100nM, 1µM, 10µM), then stained with phalloidin to visualize cytoskeletal changes. Cellular proliferation and viability were tested using a Cell Titer Glo assay. Migration changes were visualized and quantified using a Boyden migration chamber and ImageJ analysis. All treatment groups were compared to vehicle-treated controls. Results:GW525701 treatment at all concentrations caused morphologic changes consistent with senescence and apoptotic cell death. Cellular proliferation and viability were inversely correlated with GW525701 treatment concentration. Conclusions: Treatment with GW525701 may give insight into the roles of STK10, TNIK, SLK, MAP4K4, MINK, and DDR1 in TNBC survival. This information will be crucial to determining an effective means of targeting TNBC in a therapeutic manner in the future. The specific kinase(s) responsible for the phenotypic outcomes in this experiment will be elucidated in the future as well.
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    Characterization of Estrogen Receptor Mutant Breast Cancer Three-Dimensional Cell Cultures
    (2024-03-21) Wright, Jordan; Mayer, Olivia; Rinderle, Caroline; Bunnell, Bruce
    Background: Breast cancer is one of the leading types of cancer among women globally. Many primary breast cancers are estrogen receptor-positive (ER+) and responsive to anti-estrogenic therapies; however, after long-term treatment, these tumors can mutate the estrogen receptor to survive. These mutations make the tumors more triple-negative-like and, therefore, more dangerous. Triple-negative breast cancer (TNBC) has been particularly challenging to treat due to its lack of estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2) receptors. Due to the lack of treatment options, TNBC has a poor prognosis and contributes to a significant percentage of breast cancer mortalities. These ER mutants act more like TNBC, resulting in worse clinical outcomes. Current research on these ER mutants has been conducted using two-dimensional (2D) monolayer cell culture, which does not translate effectively in animal models and humans. Three-dimensional (3D) cell culture, which allows for the formation of spheroids, mimics actual tumors and provides results more consistent with actual tumor treatment in vitro. Due to the lack of research on these ER mutants in 3D culture, they first must be characterized to determine baseline gene expression and behavior. After characterization, identifying changes resulting from drug treatment will be possible. Methods: Three different MCF-7 ER+ mutant cells (D538G, E380Q, and Y537S) were seeded at a density of 3000 cells per well in a low-attachment, round-bottomed 96-well plate. After seven days of culture, spheroids were imaged. Spheroids were measured, and size differences were quantified compared to the control, wild-type, ER+ MCF-7 cell line. RNA was extracted from spheroids, quantified, and reverse transcribed to make cDNA. cDNA was used to perform qRT-PCR to determine baseline gene expression differences between ER+ control MCF-7s and ER+ mutant MCF-7 cell lines. Results: The wild-type ER+ spheres have smaller diameters, spherocity values closer to one, and are more compact. They express different levels of EMT markers from the controls, indicating alterations to signaling pathways in the mutant lines. These 3D cultures also vary in expression from the 2D cultures of the same cell lines. Conclusions: MCF-7 ER+ breast cancer cells aggregate more readily than ER+ mutants. The ER must be involved in signaling that promotes aggregation, as reduced ER signaling decreases the ability for spheroid formation. This phenomenon and the differences in gene expression may explain why these mutants tend to behave in a more triple-negative manner in humans. Cells cultured in 3D express some genes to different extents, confirming the importance of 3D culture to identify future therapies – cells behave differently in different culturing contexts, 3D being more consistent with actual tumor behavior. Therefore, characterizing ER+ mutants before beginning drug treatment studies is crucial to understanding how compounds affect cancer cells and identifying differences in different ER+ mutants to know better how to treat them in the future.
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    First-in-class Peptide Molecules Targeting the MIEN1 Cancer signaling Pathway for which no inhibitors are currently identified
    (2024-03-21) Tripathi, Amit; Ranade, Payal; Trivedi, Rucha; Vishwanatha, Jamboor K.
    Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.
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    High expression of N-acetyl transferase 9 in cholangiocarcinoma and its possible role in tumor progression
    (2024-03-21) Hoteit, Tamara; Jones, Harlan; Chaudhary, Pankaj
    Cholangiocarcinoma, CCA, is an aggressive type of liver cancer due to the scarce number of biomarkers and its resistance to anticancer drugs, leading to difficulty in its early detection. Its 5-year survival rate ranges from 2% to 24%, depending on severity and metastasis. Currently, surgery is the most effective treatment option, but only under certain criteria, such as the cancer being caught early and having not metastasized. CCA incidence is the highest in Asian countries because of the presence of a carcinogenic liver fluke. N-acetyl transferase, NAT, is an enzyme with many functions, including regulating protein stability, membrane targeting, gene silencing, and drug resistance. Different subsets of NATs are also known to be biomarkers for different types of cancer, such as colorectal, prostate, and breast cancers. According to The Cancer Genome Atlas database, NAT9, a subset of the NAT family, is overexpressed in patients with CCA; however, no studies have demonstrated its role in CCA, indicating the need for more research. In this study, we aim to assess the expression of NAT9 in CCA using cell lines and patient tissue samples through RT-qPCR, western blotting, and immunohistochemistry. This data will help us shed light on NAT9’s role in the tumorigenesis of CCA and could be a promising biomarker and therapeutic target for CCA.
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    (2024-03-21) Marikh, Morad; Brown, Ainsley; Hall, Courtney; Donkor, Michael; Garlotte, Isabelle; Subasinghe, Kumudu; Elkassih, Omar; Jones, Harlan; Phillips, Nicole
    The lung serves as a primary site for breast cancer metastasis, carrying profound implications for patient prognoses. About 60% of people diagnosed with metastatic breast cancer have lesions in either the lungs or the bones, with triple-negative breast cancer (TNBC) more likely than other types of breast cancers to metastasize to the lungs. Although current targeted chemo-radiotherapy and surgery result in higher survivorship, studies have documented that such curative treatments may also increase risk of lung metastasis. To date, the causal factors that mediate metastasis in the context of cancer treatments remain elusive. Our long-term goal is that a deeper understanding of the mechanisms that mediate relocation of breast tumor cells from its primary origin to its distal site (e.g., lung) will reveal novel complementary diagnostic and preventative treatments to improve TNBC survivorship. Exosomes, serving as tiny extracellular vesicles within tumor cells and other cells (e.g., immune cells) release diverse biomolecules have been implicated in tumor pathogenesis. Specifically, miRNAs as cargo within exosomes are known to regulate cellular function. miRNAs are small RNA molecules that can bind to messenger RNA (mRNA) and inhibit protein synthesis or promote mRNA degradation. This regulatory function allows miRNAs to modulate the expression of multiple genes involved in various cellular processes and their dysregulation has been implicated in various diseases, including cancer. The objective of this study was to determine the expression of miRNA-200b-3p and miRNA-141-5p as known regulators of lung cancer are influenced by surgical removal of a primary breast cancer. We hypothesized that miRNA-200b-3p and miRNA-141-5p mRNA expression is increased in response to surgery. Using an established model of breast cancer metastasis, exosomes were isolated from the bronchiole alveolar lavage fluid (BALF) of tumor bearing mice and mice in which primary tumors were resected compared to tumor-free mice. Results demonstrated that miRNA-200b-3p was present in both tumor-bearing and non-tumor-bearing mice. In contrast, miRNA-141-5p was not expressed in tumor-bearing, non-tumor-bearing mice, and naïve mice determined by quantitative reverse transcriptase polymerase chain reaction (qrtPCR). In conclusion, as we navigate the intricacies of miRNA dynamics in the lung microenvironment, future studies will involve broadening the miRNA panel and refining exosome recovery techniques. This strategic evolution aims to enhance sensitivity, facilitating the detection of elusive, tumor-derived exosome miRNAs. All studies have been approved by UNTHSC IACUC, approval number #2018-0031. Acknowledgement: This research is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) to Dr. Jamboor K. Vishwanatha and National Institute of Cancer Research of the Health under Award 1 P20 CA233355-01 (Vishwanatha, Jones-Project 1).