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AMA Research Challenge 2022

A Glycan-based Approach to Cell Characterization &amp; Purification

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Ricardo Correa, MD, EdD
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Stuart M. Greenstein, MD
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Stephanie Strohbeen, MD
 
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Jessica Li, MD/PhD Candidate
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Charles Lopresto, DO

### Sponsor
AMA would like to thank Laurel Road for sponsoring a $10,000 grand prize for the winner of the AMA Research Challenge. Laurel Road is not involved in the judging or scoring process to select the winner. The winner will be announced on Dec. 7 during the AMA Research Challenge finals. Payment of the grand prize will be directly from Laurel Road. See the AMA Research Challenge Official Rules for terms and conditions.

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Welcome to the largest national, multi-specialty research event featuring research from 750 medical students, residents and fellows, and international medical graduates.


A Glycan-based Approach to Cell Characterization & Purification

poster

Introduction
Achyrocline bogotensis is a member of the Asteraceae family of flowering plants. While the Asteraceae family is well known for food crops (lettuce), ornamentals (chrysanthemum), or common weeds (dandelion); A. bogotensis, itself, is know as ‘vira vira’ (the plant of life). Found in the Andean region of South America, A. bogotensis has been used ethnomedically for thousands of years. Ground into a poultice or steeped to make tea, A. bogotensis has been said to help heal wounds, fight infections, reduce inflammation, and even possess anti-neoplastic qualities. Previous studies from our lab have shown that one compound isolated from A. bogotensis, 3,5 dihydroxy-7-methoxyflavone (Achyrocline B, Flavone B, FB), is cytotoxic to colorectal and pancreatic cancer cell lines1,2. 
Surgically unresectable colorectal and pancreatic carcinomas have a high rate of mortality as current treatment options are limited. One common chemotherapeutic used to treat these cancers is 5-flurouracil (5-Fu); however, treatment typically only serves to slow the progression of the disease and comes with many side effects due to 5-Fu’s intrinsic toxicity3-6. Thus, strategies to decrease the amount of 5-Fu used in treatment as well as reduce its off-target toxicity are paramount not only to better treatment of colorectal and pancreatic carcinomas, but also to improve patient quality of life. 

Methods
Viability after treatment was measured in various colorectal and pancreatic cancer cell lines by MTT. Synergy analysis was performed using CompuSyn software. Results were supported by analysis of cellular lysates for apoptotic proteins by western. Xenograft implantation was done using HCT116 into nude mice. Mice were treated 3/week for six weeks with either saline, Achyrocline B + 5-Fu, or 5-Fu alone. Tissue was either flash frozen for western or paraformaldehyde fixed, paraffin embedded, and then analyzed using H&E. All statistical analyses was performed using GraphPad Prism.

Results
Synergy analysis revealed that cotreatment with Achyrocline B and 5-Fu was synergistic and as efficacious as a dose of 5-Fu four-fold higher in multiple colonic and pancreatic cell lines. These results were supported by western analysis of apoptotic proteins. Off target toxicity models showed an opposite profile in which less loss of viability and apoptosis was seen. In our xenograft model, Achyrocline B was shown to increase apoptosis in tumors leading to degeneration of the outer tumoral boundary. Furthermore, in 5-Fu treated animals it was found that Achyrocline B provided protection on both a histological and cellular level. 

Conclusion
Our studies show that Achyrocline B efficiently allows for 5-Fu dose reduction and has dual actions: 1) It works synergistically with 5-Fu to kill cancer cells in vitro and in vivo and 2) protects tissue in vivo from the toxicity of 5-Fu. These results show promise for a new adjuvant therapy for colorectal and pancreatic carcinomas that not only reduces tumor progression, but more importantly has the potential to improve patient quality of life. 


Achyrocline B (3,5 dihydroxy-7-methoxyflavone) synergizes with 5-fluorouracil (5-Fu) allowing for 5-Fu dose reduction and reduced off-target toxicity in the treatment of colonic and pancreatic cancers

Background
In recent years, glycobiology has garnered increasing interest in relation to oncology research. Glycans are complex carbohydrates on the surface of most eukaryotic cells and have been found to contribute to cancer tumorigenesis and metastasis. Patterns of aberrant glycan expression, such as increased sialylation and fucosylation, have been proposed as potential novel biomarkers of cancer. However, to our knowledge, there is little research examining the differences in glycosylation between malignant and surrounding benign tissues that can be applied in a clinical setting. This study examines the N-linked glycan expression and composition in samples of cancerous and surrounding healthy tissue of breast cancer patients, with the aim of establishing signature glycosylation patterns between the two tissue types. 

Methods
Thirty-nine samples of malignant and surrounding benign tissue from breast cancer patients were collected from the Prisma Health Biorepository. The samples included specimens from breast cancer staged I through IV at the time of the patients’ diagnosis. The samples were analyzed using MALDI-TOF mass spectroscopy for the presence of glycans by Emory Glycomics and Molecular Interactions Core. The known masses of sixty-one unique glycans were then compared to the spectra of the samples analyzed to determine the type of glycans present. The relative amount of each glycan present was determined using the area under the curve in the spectral data.

Results
The means of the area under the curve for high-mannose glycans in the malignant and normal tissue samples were 247.18 and 163.6 respectively. The t-test reveals a significant difference in the expression of high-mannose glycans between the two tissue types (p-value, 0.0138).

Conclusion
The results of this data confirm a higher expression of high-mannose glycans to be a potential biomarker for malignant tissue in breast cancer. Future research could include expanding the sample size and comparing the glycan composition of the samples to a library specific for high-mannose type glycans, with the goal of establishing a signature high-mannose expression pattern. This could lead to important clinical applications such as being a helpful distinction in obtaining negative margins during tumor resection, as well as providing a more refined diagnostic method and more informed treatment decisions. 




Analysis of Glycan Expression in Malignant versus Adjacent Benign Tissue in Breast Cancer Patients

In our previous work, we identified clustering of kidney cancers based on differential metabolic pathway activation, and an association between metabolic clustering and survival in renal cell carcinoma. Here we further study clear cell renal carcinoma, papillary cell renal carcinoma and chromophobe renal cell carcinoma by applying machine learning models to pinpoint specific metabolic reactions, and their master regulators, that best explain differences in survival in these cancers. We constructed a cohort of 941 patients in The Cancer Genome Atlas (TCGA) diagnosed with kidney cancer (project codes KIPR [papillary cell RCC], KIRC [clear cell RCC] and KICH [chromophobe RCC]). We used tumor RNA-Seq data to construct reaction activity scores (RAS) using metabolic genes according to the HMRcore rules and taking into account enzymatic subunits and multiple different genes coding for proteins that catalyze the same reaction. We then used a machine learning model to estimate survival as a function of metabolic pathways, age and gender. By using Cox regularization with elastic-net penalty we were able to simultaneously perform variable selection and estimation of survival in presence of a large number of regressors. Using master regulatory analysis, we estimated activity of key transcription factors and then used a multinomial model with elastic net penalty to identify those key transcription factors that have the greatest impact on dysregulation of tumor metabolic pathways in renal cell carcinomas. Using glucose transport as a known hallmark of renal cell carcinoma, we identified 19 metabolic reactions that are better predictors of survival than glucose transport in our machine learning model. The metabolic reactions we identified fit into larger categories that include folate and protein metabolism, as well as beta-oxidation. These are consistent with metabolic changes induced by mTOR, and it is notable that inhibitors of mTOR are already used in the clinic for treatment of renal cell carcinoma. Our research raises the question of whether effectiveness of mTOR inhibitors can be increased by amplifying the effects of mTOR inhibition on oncogenesis through targeting of related master regulators of metabolism.

Analysis of Metabolic Profile and Survival Data in Renal Cancer

Multiple myeloma (MM) is a plasma cell malignancy that grows in the bone marrow and is characterized by severe bone destruction. The growth of MM cancer cells in the bone marrow increases osteoclasts, bone-resorbing cells, which create debilitating bone lesions. MM cells also decrease osteoblasts, bone-forming cells, which reduces bone formation. Despite recent advances in anti-MM therapy, MM patients frequently relapse and exhibit high fracture incidence even during disease remission. Our lab showed that inhibition of Notch, a pathway regulating cell-to-cell communication in the tumor niche, with a bone-targeted Notch inhibitor (BT-GSI) decreases tumor growth and bone resorption without inducing toxicity. We also found that pharmacologic inhibition of Sclerostin (Scl-Ab), a potent inhibitor of Wnt signaling and osteoblast function, increases bone formation without affecting tumor growth. In this study, we hypothesized that co-administration of BT-GSI and Scl-Ab simultaneously decreases tumor burden and improves bone health by inhibiting bone resorption and increasing bone formation. 6-week-old immunocompetent mice were injected intratibially with 105 murine 5TGM1 myeloma cells. After four weeks, mice with MM were randomized to groups receiving vehicle, Scl-ab (100mg/Kg/1x/wk), BT-GSI (2.5mg/Kg/3x/wk), or Scl-ab+BT-GSI combined for four additional weeks. Longitudinal micro-CT was performed every four weeks to assess bone mass and microarchitecture. Alizarin Red and Calcein fluorescent dyes were injected 5 and 2 days, respectively, before sacrifice. Bone dynamic and static histomorphometry analyzed fluorescent labels and osteoclasts present via TRAP staining using OsteoMeasure. Mice treated with Scl-Ab therapy exhibited similar tumor burden levels than mice receiving vehicle injections. In contrast, mice treated with BT-GSI, either alone or in combination with Scl-ab, exhibited decreased tumor burden compared to vehicle-treated mice. Mice receiving vehicle injections had decreased bone mass compared to control naive mice. All three therapies significantly increased bone mass compared to vehicle mice. There was no organ toxicity with any of the therapies. We performed static and dynamic histomorphometric analysis to determine the mechanism underlying the skeletal protective effects. Vehicle-treated mice had decreased bone formation rate (BFR) compared to naïve mice, although not statistically significant. Scl-ab therapy increased BFR by 28%, bringing it back to naïve levels. BT-GSI therapy did not affect BFR, which remained decreased compared to naïve mice. Co-administration of BT-GSI and Scl-ab also restored BFR to naive mice levels. Vehicle-treated mice had a five-fold increase in osteoclast number corrected by bone surface (Oc.N/BS) compared to naïve mice. Scl-ab and BT-GSI induced a 39% and 75% decrease in Oc.N/BS compared to MM-bearing mice receiving vehicle, respectively. Co-administration of BT-GSI and Scl-ab also resulted in a 75% reduction in Oc.N/BS compared to MM mice, bringing Oc.N/BS back to levels found in naïve mice. BT-GSI + Scl-ab combination therapy decreases tumor burden and improves bone health by decreasing osteoclasts and increasing the bone formation rate. BT-GSI + Scl-ab combination therapy is more effective than these therapies alone in stopping tumors and repairing damaged bone. Because this bone-targeted multimodal therapy lacks toxicity, it provides a new therapeutic avenue to control MM growth and improve bone health in MM patients.

Bone-targeted combination therapy decreases tumor progression and restores bone mass by increasing bone formation in mice with established multiple myeloma

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Non-small cell lung cancer is the most prevalent and includes adenocarcinoma (AD), and squamous cell lung cancers. The unique cellular biology of these cancers could potentially allow for serological detection and identification via tissue specific protein markers. Galectins are a group of β-galactoside binding proteins which are known to be alternatively expressed in cancers and are being studied for their biomarker potential. Galectin-7 is expressed in epithelial tissues as well as in all layers of the epidermis as it is normally highly expressed by squamous cells. We evaluated the potential for galectins to be used as a serum biomarkers and delineators of two non- small cell lung cancer subtypes.
The serum concentrations of galectin-7 were measured in 85 lung cancer patients using ELISA. Forty-eight samples were from adenocarcinoma patients and 37 were from squamous cell carcinomas patients. The serum galectin-7 concentrations in cancer patients were compared to healthy controls and by their histological subtypes. Oneway analysis was performed using Tukey-Kramer HSD for pairwise comparisons.
Galectin-7 concentrations were significantly elevated in serum samples from squamous cell lung cancer patients (mean, 2.45 ng/mL) relative to healthy controls (N, 26; mean, 1.16 ng/mL; p- value, 0.0010) and relative to adenocarcinoma lung cancer patients (mean, 1.47 ng/mL; p-value, 0.0040).
These results show that serum galectin-7 is increased in patients with squamous cell lung cancer compared to adenocarcinoma lung cancer. Further benchwork would include (1) measuring serum concentrations of galectin-7 by stage of squamous cell lung cancer, (2) identifying concentrations of serum galectin-7 in other lung cancer subtypes, and (3) in other non-pulmonary squamous cell carcinomas such as skin, cervical, and head and neck. Potential clinical applications of this work would include the measurement of galectin-7 in blood samples of cancer patients to aid in cancer screening and tumor type identification.

Comparison of Galectin-7 Serum Concentrations in Squamous Cell and Adenocarcinoma Lung Cancers

Concurrent malignant and benign nephrosclerosis in an elderly cadaver with previously diagnosed cerebral atherosclerosis

Background: 4-1BBL is a co-stimulatory molecule that is important for T-cell expansion and
induction of robust CD8+ T-effector and memory responses, critical for tumor rejection. The natural 4-1BBL is a trimeric transmembrane protein that has no activity in soluble form. To overcome this limitation, a unique soluble form of 4-1BBL was developed by linking core streptavidin (SA) to the extracellular domain of murine 4-1BBL (SA-4-1BBL). This chimeric protein forms oligomers that have pleiotropic effects on cells of innate, adaptive, and regulatory immunity. However, production and purification of SA-4-1BBL is cost- and time-intensive. Therefore, development of an expression platform that is more efficient, translational, and augments anti-tumor efficacy of SA-4-1BBL will have a significant impact for clinical development of this immune adjuvant. Thus, we developed a novel oncolytic adenoviral system to deliver SA-4-1BBL to solid tumors.
Methods: The AdenoQuick system was used to construct OAds expressing SA-4-1BBL or SA (control). Transgene expression was confirmed by western blot and SA-4-1BBL biological function was evaluated by splenocyte proliferation assay in vitro. The OAdSA-4-1BBL and
Ó 2022 American Medical Association. All rights reserved.
OAdSA-mediated cancer killing effect was evaluated by crystal violet staining, the activation of autophagy-mediated immunogenic cell death was assessed by detection of autophagy markers LC3-I, LC3-II and p62. The damage-associated molecular patterns molecules adenosine triphosphate, high-mobility group box 1, and calreticulin were evaluated by luminescence, ELISA, and flow cytometry, respectively. Tumor expression of SA, SA-4-1BBL, and Hexon was evaluated by immunohistochemistry. The OAdSA-4-1BBL therapeutic efficacy was evaluated in C57BL/6 mice-bearing subcutaneous TC-1 lung tumor.
Results: We successfully constructed OAds expressing SA or SA-4-1BBL. We demonstrated that SA-4-1BBL can produce biologically active oligomers, selectively induces autophagy- mediated immunogenic cell death in murine and human lung cancer cell lines with little cytotoxicity to normal cell lines. The transgene expressions within the subcutaneous tumor were confirmed and OAdSA-4-1BBL shows greater anti-tumor efficacy than control viruses in a TC-1 tumor mouse model.
Conclusion: Overall, our data suggests that OAd expressing the immune checkpoint stimulator SA-4-1BBL may represent alternative therapeutic strategy against lung cancer, which could be used alone or in combination with immune checkpoint inhibitors, or other approaches aimed at enhancing the anti-tumor immune response.

Development and characterization of an oncolytic adenovirus armed with the immune checkpoint stimulator SA-4-1BBL

Effect of Hesperetin against DMH induced toxicity in the colon of Wistar rats

Background
Glioblastoma (GBM) represents the most common primary malignant brain tumor in adults, with a median survival rate of less than two years. GBM presents with therapeutic resistance and high rates of recurrence, largely attributed to the presence of glioblastoma stem cells (GSCs). GSCs are frequently located in specific niches, including a hypoxic niche and a normoxic, perivascular niche. Targeting GSCs is a potential therapeutic avenue for treating GBM, but the details of GSC function in hypoxic conditions remains poorly understood. Further, most gene expression findings are limited to epigenetic or transcriptional regulation assays, overlooking the importance of the translatome. This study expands on conclusions made using steady-state mRNA levels by including ribosome profiling to evaluate translational regulation mechanisms. 

Methods
We selected two patient-derived GSC lines (GSC 215, GSC 316) and cultured them in both hypoxic (5% O2) and normoxic (21% O2) conditions. RNA-seq libraries were prepared in parallel with RIBO-seq libraries for the lines in both conditions, with biological replicates for each treatment group, after which Illumina sequencing was performed. Raw sequencing reads were assessed for quality. Single-end reads were aligned and counted using command line versions of STAR, BEDTools, and SAMTools. Differential changes in ribosome-protected fragments (∆RPF) and changes in transcript levels (∆RNA) were calculated using DESeq2. GO term analyses for transcripts was performed with Reactome using a hypergeometric statistical test and BH FDR correction.

Results
Comparing ∆RNA and ∆RPF levels between hypoxic and normoxic GSCs reveals 9 functional clusters of genes. These categories indicate genes which are post-transcriptionally controlled in 3 manners: translationally suppressed (28.19%), translationally upregulated (32.87%), or concurrently translated (38.93%). Altogether, more than 60% of mRNA show marked regulation on the translational level.

Significant enrichment of genes in the NOTCH signaling pathway (p = 1.09e-02), WNT signaling pathway (p = 1.37e-04), and RHO GTPase cycle (p = 2.37e-07) are present among the transcripts that experience translational upregulation in hypoxic conditions.

Conclusion
This study may lead to the identification of potential therapeutic targets as evidenced by their function as regulatory hubs in cellular networks responsible for hypoxic adaptation. These observations are largely undetected by RNA-seq measurements.


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Achyrocline B (3,5 dihydroxy-7-methoxyflavone) synergizes with 5-fluorouracil (5-Fu) allowing for 5-Fu dose reduction and reduced off-target toxicity in the treatment of colonic and pancreatic cancers

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