Trainee Talks

10-minute presentation format

Analine Aguayo (undergraduate student), CSU Northridge - Biology

Secretomes from Metastatic Breast Cancer Cells Enriched for a Prognostically Unfavorable LCN2 Axis Induce Differential Stromal Remodeling within the Lung and Brain

Metastatic spread of tumors accounts for over 95% of cancer-related deaths. Before cancer cells can survive in distant microenvironments, these tissues must be primed to acquire tumor-permissive properties. The primary tumor communicates with the premetastatic niche by releasing factors that recruit cells, such as mesenchymal stem cells (MSCs) and macrophages, to be reprogrammed and support this tumor-permissive environment. However, the cell-cell communication that occurs during premetastatic niche priming is poorly understood. To address this, we employed in vivo and in vitro methods to evaluate how secretomes from metastatic/non-metastatic mouse and human breast cancer cells (BCCs) may promote MSC recruitment and macrophage polarization. We show that secretomes from metastatic BCCs remodel lung and brain tissues in a manner that may support cancer cell seeding and expansion, with evidence of MSC-macrophage cross-talk only being observed in the brain. Furthermore, we show that secretomes from BCC-educated MSCs or macrophages/monocytes increase protumorigenic cell states of macrophages/monocytes or MSCs, respectively. Finally, analysis of secreted factors differentially produced by metastatic BCCs revealed an LCN2 axis that predicts poor survival in breast cancer patients. These results indicate that cell-cell crosstalk may mediate reprogramming of the premetastatic niche and justify developing targeted therapeutics to inhibit this cross-talk.

Erick Mitchell-Velasquez (undergraduate student), CSU Northridge - Biology

The Role of E4BP4 in Apoptosis of Human Leukemia Cells in Culture

Apoptosis is a physiological and genetically controlled process of cell death important for development and cellular homeostasis. Several diseases can be attributed to an imbalance between cellular proliferation and attrition, brought on by inadequate or excessive apoptosis. In cancer, a lack of apoptosis causes excessive accumulation of unwanted cells. Apoptosis is primarily regulated via altered expression of key genes and modulation of signal transduction pathways. Our laboratory focuses on understanding the early apoptosis events triggered by chemotherapeutic agents in cell culture models of leukemia. Previous studies have shown that the E4BP4 (adenovirus E4 binding protein 4) gene in upregulated in correlation with apoptosis. E4BP4 upregulation has been implicated in induction of the pro-apoptotic BIM protein, a member of the Bcl-2 family of proteins. In studies presented here, we have utilized CRISPR-mediated genomic editing technology to knock down E4BP4 expression. Three clones with reduced E4BP4 expression were analyzed for their response to Dexamethasone (Dex), a synthetic glucocorticoid and anti-leukemic agent. While E4BP4 knockdown did not alter the ability of cells to undergo apoptosis in response to Dex, two of the three clones showed a reduction in the extent of BIM upregulation. Residual E4BP4 expression in our knock down clones may be sufficient to evoke an apoptotic response. Further efforts are underway to understand downstream pathways regulated by E4BP4.

Brandy White (graduate student), CSU Fresno - Chemistry and Biochemistry

Humanization of a MUC16 Specific Monoclonal Antibody for the Treatment of Pancreatic Cancer

Pancreatic ductal adenocarcinoma is a highly lethal form of cancer. The near-death sentence is associated with the failure of traditional therapeutics. The clinical success of therapeutic antibodies for cancer treatment is now well established, however no approved antibody therapies for pancreatic cancer exist. Here we report the humanization of a therapeutic antibody, which in mouse models halted the proliferation and metastasis of pancreatic cancer. The antibody binds MUC16, an overexpressed and abnormally glycosylated membrane-bound protein. The antibody was humanized by grafting the antigen-binding CDR loops onto a human germline antibody scaffold. Venier zone residues from the mouse sequence predicted to be important for the structure of the CDR loops were conserved. Transient transfection of the humanized antibody resulted in a yield of 1 mg per ml of media. ELISA binding assays demonstrated that the humanized and parent mouse antibodies bound MUC16 with a comparable affinity. Epitope mapping revealed that the antibody bound an SEA domain within the tandem repeat region of MUC16. Finally, a high-resolution X-ray structure of the humanized antibody Fab region revealed an extended light chain CDR1 and binding pocket for the MUC16 SEA domain. The structure illuminated the importance of Vernier zone residues in maintaining CDR loop conformations. The results presented here pave the way for future engineering of this novel antibody for the treatment of pancreatic cancer.

Katherine Reil (graduate student), SDSU - Biology

VAX014, a novel immunotherapy, inhibits orthotopic bladder cancer in preclinical murine models

Immunological recognition to cancer is a critical feature in successful treatment, allowing the host’s immune system to work as an effective defense. Vaxiion Therapeutics has developed a non-infectious bacterial immunotherapy known as minicells that encapsulate and deliver a wide array of anti-tumor defense molecules in addition to potentially promoting anti-tumor immunity. In collaboration with SDSU, minicells called VAX014, have been tested in the orthotopic MB49 bladder cancer model in C57BL/6 mice to determine if VAX014 is applicable to the treatment of non-muscle invasive bladder cancer (NMIBC) in humans. Complete tumor remission was observed in 70% of VAX014 treated animals (p=0.01) in an immediate post-operative model where a single dose of VAX014 was administered immediately following MB49 bladder implantation. To mimic early stage treatment, VAX014 was delivered 24 hours after tumor implantation on a q7dx6 schedule with a 60% survival increase (p<0.001). Moreover, VAX014 was tested in an aggressive model with treatment beginning on day 7 post MB49 installation and overall survival of treated animals was significantly extended beyond the control group (p=0.02). In all instances, animal survivors were shown to reject orthotopic tumor re-challenge (p=0.0001) thereby indicating an immune-mediated anti-tumor response and the potential to prevent metastasis. These preclinical data make VAX014 a promising immunotherapy for the NMIBC patient community.

Katherine Jensen (graduate student), Chico State - Nutrition and Food Science

Effects of Sulforaphane on proliferation and invasion capacity of triple negative breast cancer cells exposed to tumor-associated macrophages

Triple negative breast cancer (TNBC) is characterized by high proliferation rates and an increased likelihood of invasion and metastasis. Tumor cells and cells located in the tumor microenvironment (TME), such as tumor associated macrophages (TAMs), interact through signals (e.g., cytokines) to promote cancer progression. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables that has been shown to reduce tumor growth in animal models and decrease invasion markers in TNBC cells grown in isolation. However, presence of TAMs in the TME can enhance tumor cell proliferation and invasion. The objective of this study was to determine if SFN can reduce proliferation and invasion of TNBC cells grown under the influence of TAM's. A conditioned-media approach was used to model non-contact interactions found in the TME using THP-1 human monocytes and MDA-MB-231 human TNBC cells. THP-1 cells were differentiated in 50% (v/v) cancer-conditioned media to create TAMs. MDA-MB-231 cells were grown in 50% (v/v) TAM-conditioned media for the remaining experiments. Cells were then treated with SFN (10 μM) or DMSO vehicle control. Proliferation was measured by an MTT-based colorimetric assay. Invasion capacity was measured by a Transwell Invasion assay. ANOVA and t-tests were used to determine statistical differences. Significant reduction in cell proliferation for the TAM-educated MDA-MB-231 cells after 24 hours (p=0. 0132) and 48 hours (p=0. 0190) of SFN treatment.

Arabo Avanes (graduate student), CSU Los Angeles - Chemistry and Biochemistry

The Role of t-Darpp in Breast Cancer Cell Migration

t-Darpp is overexpressed in breast cancers at a 50% frequency and has been shown to confer trastuzumab-acquired resistance in HER2-type breast cancer. A key phosphorylation site at Thr39 is critical for resistance and has been shown to be a substrate for CDK1 and CDK5. Breast cancers become particularly difficult to treat when they become metastatic. Emerging evidence in lung cancer cells indicates that t-Darpp promotes cancer cell migration - a characteristic of metastasis. To assess whether Thr39 plays a role in t-Darpp-mediated cancer cell migration, a wound healing assay with t-Darpp-expressing breast cancer cells was utilized. Isogenic breast cancer cell lines overexpressing wild-type t-Darpp, Thr39Ala mutant t-Darpp, and the Thr39Asp phosphomimetic mutant t-Darpp were each assessed for cell migration. Preliminary results of our study and possible mechanisms to account for these outcomes will be presented.

5-minute presentation Format

Ryan Meza (undergraduate student), CSU Northridge - Engineering

Effects of DOD 3D Bioprinting on the Formability of Microspheres

The growing number of people in need of organ transplants has increased steadily over the past few decades; however, the number of organ donors has not seen a large increase in about 10 years. As organ donor waitlists continue to increase to an all-time high, 3D Bioprinting has emerged as a promising technological advancement to possibly solve this problem. Tissue engineering through freeform fabrication has provided an opportunity to have an unlimited supply of biocompatible tissues and organs using one's own cells. In this study, the compatibility of various bioinks were examined using a Drop-on-Demand, piezoelectric inkjet 3D bioprinter. We tested the properties of various bioinks in relation to this device to determine optimal bioink composition and printing parameters. To test various bioinks under extreme conditions, hydrogels were extruded on a MicroFab PH-46 (MicroFab, Plano, TX) micro dispensing subsystem. This system consists of a MicroFab Jet Driver responsible for controlling the DOD pulse, a MicroFab pneumatic controller used to adjust the back pressure, an 80um piezoelectric micronozzle used to extrude bioinks, and an imaging camera STC-MB33USB (SENTech America, Carrollton, TX) to observe the droplet formation process. Biomaterials used in this experiment include sodium alginate (0.5% w/v, 0.75% w/v, 1% w/v, and 1.25% w/v) and sodium alginate (0.5% w/v) with gelatin (0.1% w/v).

Andrew Macias (undergraduate student), SDSU - Biology and Bioinformatics

Onco-GPS map depicting the cellular states of Neuroblastoma cell line response to 13-cis-retinoic acid

The purpose of this study is to identify the therapeutic response of 13-cis-retinoic acid (13-cis-RA) on Neuroblastoma cell lines and to develop an OncoGenic Positioning System (Onco-GPS) map describing the drug response. Neuroblastoma is a pediatric cancer that can arise anywhere on the sympathetic nervous system and is a leading cause of death from pediatric cancer for children between the ages of 1 and 5. 13-cis-RA is a retinoid used to control cell growth, cell differentiation, and cell death during embryonic development. The Neuroblastoma cell lines used in this study were SK-N-SH, SK-N-BE(2), SK-N-AS, IMR32, CHP-134, Kelly, LAN-1, and NGP. These cell lines were treated with 13-cis-RA and were placed in a live-cell analysis imaging system that captured images every 6 hours over a span of 5-7 days. After the analysis was done, it was determined that SK-N-BE(2), SK-N-SH, CHP-134, LAN-1, and NGP are 13-cis-RA sensitive, while SK-N-AS and IMR32 are 13-cis-RA resistant; and the Kelly cell line only responded to 13-cis-RA at a higher concentration of cells/well. The drug response for SK-N-SH, SK-N-BE(2), SK-N-AS, and IMR32 can be seen on the Onco-GPS map. The map highlights the effect of adding 13-cis-RA to the sensitive cell lines by the change in cellular states. Cellular states are defined as grouping samples together based on similar gene expression. From this study, Neuroblastoma cell lines are able to show responsiveness to 13-cis-RA, as well as other therapeutic drugs.

Yesenia Avitia (undergraduate student), SDSU - Public Health

HPV Vaccine Prevalence in Pediatric Oncology Survivors

HPV vaccination reduces the risk of cervical cancer. HPV vaccination rates are low among teens. Pediatric oncology survivors have a greater risk of acquiring this infection increasing the risk of HPV-related cancers. Limited information exists about HPV vaccination behavior in children with cancer. This study assessed the proportion of pediatric oncology patients who received at least one HPV vaccination and evaluated differences in HPV vaccination by health literacy level and Hispanic ethnicity. Parents or guardians of childhood cancer survivors were administered questionnaires to assess their child’s HPV vaccination status, health literacy, and ethnicity. Parental health literacy was measured by Newest Vital Sign and categorized into: high likelihood of limited health literacy, possibility of limited health literacy, and adequate health literacy. Of the 148 parents, 27.7% reported their child had at least one HPV vaccine. 15% of parents or guardians who had a high likelihood of limited literacy reported a HPV vaccination. HPV vaccination was reported by 37% of parents or guardians with possibility of limited health literacy; whereas those with adequate health literacy, 28% reported their child had been vaccinated. HPV vaccination prevalence was lower in Hispanic (25.0%) than non-Hispanic (31.2%) childhood cancer survivors. Interventions to increase HPV vaccination in pediatric cancer survivors are warranted, especially in those who are Hispanic or with low health literacy.

Benjamin Cordova (graduate student), CSU Northridge - Chemistry and Biochemistry

Inhibition of Glyoxalase 1 results in marked increase in intracellular glutathione and reduced cell viability in breast and prostate cancer cell lines

Glyoxalase 1 (Glo 1) predicts for poor prognosis in prostate cancer and is over-expresed in breast tumor tissue relative to pair-matched, adjacent tissue. Glyoxalase 1 and 2 is a highly conserved system responsible for the detoxification of reactive carbonyl species such as methylglyoxal and glyoxal. Both can form adducts with cellular nucleophiles such as nucleic acids, amino acids, and lipids, and lead to cellular dysfunction and cell death. The main objective of our study is to investigate Glo 1 inhibition by S-p-bromobenzylglutathione cyclopentyl diester (BBGC). We have determined IC50 values in a panel of prostate and breast cancer cell lines using the MTT assay. All IC50 values fell within 9-20uM. We used a modified version of the Tietze method for quantification reduced and oxidized glutathione (GSH and GSSG) in BBGC treated and untreated cells. Treatment of cells at the IC50 concentration elevates intracellular GSH by 10-50% for all except for the DU 145 prostate cell line. We isolated genomic DNA to quantify the levels of the DNA adduct, (R)-/(S)-N2-(1-carboxyethyl)-2’-deoxyguanosine ((R)-/(S)-CEdG) in both BBGC treated and untreated cells. We hypothesize that BBGC treatment will result in an elevated level of (R)-/(S)-CEdG adducts versus untreated cells. Taken together, metabolically targeting Glo 1 may represent a novel and viable therapeutic strategy in TNBC and CRPC. [This work is supported in part by a CSUPERB New Investigator Award to D.T.]