vestigate Their Tumorigenic Potential in High-Grade Serous Ovarian Cancer

Authors:

Brianna Carbajal, Xingeng Zhao, Cheng Wang

Date Created:

2025-01-01

Course Title:

Professor:

Not specified

About Paper:

High-grade serous ovarian cancer (HGSOC) is the most lethal immortalization. form of epithelial ovarian cancer. While most models focus on the fallopian tube epithelium, recent work suggests that ovarian If cells demonstrate immortalization, I will transplant them into immunocompromised mice to assess their ability to form granulosa cells (GCs) may also give rise to malignancy. This tumors. Tumors will be harvested for histological analysis and projectinvestigateswhetherdisruptingkeytumorsuppressorgenes RNA sequencing to evaluate HGSOC-like features and pathway in GCs can drive early tumorigenic transformation. activation. I collected granulosa cells from superovulated Cas9-expressing By modeling the effects of defined genetic alterations in ovarian mice and electroporated them with sgRNAs targeting BRCA1, GCs, this project aims to clarify whether these cells can serve BRCA2, TP53, and PTEN (B1B2PT) or Lats1 and Lats2 (L1L2). These combinations are frequently mutated in HGSOC and have as a cell-of-origin for HGSOC. The findings may reshape our demonstrated oncogenic potential in vivo. Electroporated cells understanding of ovarian cancer initiation and offer a new platform forstudyingtumorevolutionandtherapeuticvulnerabilityinanon- were cultured and monitored for sustained proliferation, extended epithelial ovarian context. passage capability, and escape from senescence as evidence of Investigating S. aureus Exposure in Food Allergy Sensitization via Bacterial Protease V8 Jessie Ma, Antonia Wallrapp, Isaac Chiu Harvard College | Eliot House | Molecular and Cellular Biology | 2028 Food allergy is a harmful immune reaction against food allergens, contributes to sensitization to food antigens, particularly through with symptoms ranging from hives and diarrhea to life-threatening V8 protease. Preliminary experiments in a mouse model of anaphylaxis. Itaffects11%ofadultsand8%ofchildrenintheU.S., epicutaneous S. aureus exposure support this hypothesis, although with prevalence rising in industrialized countries. Food allergy specific mechanisms have not yet been determined. development occurs in two phases: antigen sensitization and This study aims to investigate the mechanisms behind how V8 challenge. During sensitization, antigen-specific IgE antibodies protease promotes sensitization to food allergens at the skin. are produced and bind to mast cells. Upon subsequent challenge with the antigen, mast cells release inflammatory mediators, We utilize a mouse model of allergen sensitization with food causing anaphylaxis. antigen Ovalbumin (OVA). Repeated topical application of OVA and intradermal injections of V8 protease will be performed to While the intestine has been considered the focus of allergen induce sensitization. Protein levels of alarmins IL-33, TSLP, and sensitization, recent studies suggest that sensitization can also IL-25 and antibodies IgG1 and IgE will be determined in skin occur at the skin. In the “atopic march,” many patients who and blood samples using ELISA. Immune cell profiling on skin have atopic dermatitis will subsequently develop food allergies. tissue and draining lymph nodes will be assessed through flow Research from the Chiu lab demonstrated that Staphylococcus cytometry. Understanding and characterizing mechanisms of V8- aureus, a human pathogen commonly found in atopic skin lesions, mediated food allergy sensitization may reveal novel therapeutic induces itch by directly acting on sensory neurons via V8 protease targets and provide insight into the skin-to-gut axis in food allergy (sspA), exacerbating skin damage. We hypothesized that S. aureus development. 142 Program for Research in Science and Engineering Reprogramming of Granulosa Cells via BTP and L1L2 Gene Disruption to Investigate Their Tumorigenic Potential in High-Grade Serous Ovarian Cancer Devin Morales, Jason Yu, Sam Van OOijen, Doeke Hekstra Harvard College | Quincy House | Chemical & Physical Biology and Applied Math | 2028 Aneuploidy, the gain or loss of chromosomes, is a leading improving brightness and photostability for clearer visualization of cause of genetic disorders such as Down syndrome, cancer, and chromosomal behavior. In the final step, I will select for a third other developmental conditions. My project aims to improve plasmid containing the plexAbox promoter, lacO, centromere 3, the effectiveness of a bio-orthogonal mechanism for inducing andkanamycinresistance. Inducingtranscriptionatthecentromere aneuploidy in Saccharomyces cerevisiae, enabling us to study disrupts kinetochore attachment, causing missegregation during this condition using Raman spectroscopy. The auxotrophic yeast telophase as the mitotic spindles retract—resulting in aneuploid strain used allows for straightforward selection in downstream daughtercells. ThelacI-lacOsystemwillbeusedtovisualizethese experiments. events, with lacO positioned downstream of centromere 3 and lacI tagged with mNeonGreen. The first phase involved amplifying a plasmid containing mCherry and a kanamycin-resistance gene using forward and reverse The project is ongoing. Future plans include completing primers with homologous tails flanking the HTA2 sequence, the transformations, analyzing different strains using Raman enabling targeted insertion into the genome. We then performed spectroscopy—a method that measures the vibrational energy of a transformation on YPD plates containing kanamycin to select for molecules through light energy—under varying environmental integration. This transformation allows for visualization of the cellnditions, and applying the mechanism to different chromosomes cycle, as HTA2 expression levels vary with cell cycle progression. to study broader effects. Ultimately, this project seeks to refine an Next, I plan to introduce another plasmid containing a synthetic existingmethodofinducinganeuploidybyincreasingitsefficiency and enabling clearer, real-time visualization. By integrating transcription factor with a plexAbox promoter recognition site, an this system with Raman spectroscopy, we hope to offer a more estrogen receptor, and an activation domain. This construct also quantitative and detailed approach to studying the molecular and includes the lacI repressor, a functionalHIS gene, and GFP. I will thenperformaGibsonassemblytoreplaceGFPwithmNeonGreen, structural dynamics of aneuploidy.

Abstract:

High-grade serous ovarian cancer (HGSOC) is the most lethal immortalization. form of epithelial ovarian cancer. While most models focus on the fallopian tube epithelium, recent work suggests that ovarian If cells demonstrate immortalization, I will transplant them into immunocompromised mice to assess their ability to form granulosa cells (GCs) may also give rise to malignancy. This tumors. Tumors will be harvested for histological analysis and projectinvestigateswhetherdisruptingkeytumorsuppressorgenes RNA sequencing to evaluate HGSOC-like features and pathway in GCs can drive early tumorigenic transformation. activation. I collected granulosa cells from superovulated Cas9-expressing By modeling the effects of defined genetic alterations in ovarian mice and electroporated them with sgRNAs targeting BRCA1, GCs, this project aims to clarify whether these cells can serve BRCA2, TP53, and PTEN (B1B2PT) or Lats1 and Lats2 (L1L2). These combinations are frequently mutated in HGSOC and have as a cell-of-origin for HGSOC. The findings may reshape our demonstrated oncogenic potential in vivo. Electroporated cells understanding of ovarian cancer initiation and offer a new platform forstudyingtumorevolutionandtherapeuticvulnerabilityinanon- were cultured and monitored for sustained proliferation, extended epithelial ovarian context. passage capability, and escape from senescence as evidence of Investigating S. aureus Exposure in Food Allergy Sensitization via Bacterial Protease V8 Jessie Ma, Antonia Wallrapp, Isaac Chiu Harvard College | Eliot House | Molecular and Cellular Biology | 2028 Food allergy is a harmful immune reaction against food allergens, contributes to sensitization to food antigens, particularly through with symptoms ranging from hives and diarrhea to life-threatening V8 protease. Preliminary experiments in a mouse model of anaphylaxis. Itaffects11%ofadultsand8%ofchildrenintheU.S., epicutaneous S. aureus exposure support this hypothesis, although with prevalence rising in industrialized countries. Food allergy specific mechanisms have not yet been determined. development occurs in two phases: antigen sensitization and This study aims to investigate the mechanisms behind how V8 challenge. During sensitization, antigen-specific IgE antibodies protease promotes sensitization to food allergens at the skin. are produced and bind to mast cells. Upon subsequent challenge with the antigen, mast cells release inflammatory mediators, We utilize a mouse model of allergen sensitization with food causing anaphylaxis. antigen Ovalbumin (OVA). Repeated topical application of OVA and intradermal injections of V8 protease will be performed to While the intestine has been considered the focus of allergen induce sensitization. Protein levels of alarmins IL-33, TSLP, and sensitization, recent studies suggest that sensitization can also IL-25 and antibodies IgG1 and IgE will be determined in skin occur at the skin. In the “atopic march,” many patients who and blood samples using ELISA. Immune cell profiling on skin have atopic dermatitis will subsequently develop food allergies. tissue and draining lymph nodes will be assessed through flow Research from the Chiu lab demonstrated that Staphylococcus cytometry. Understanding and characterizing mechanisms of V8- aureus, a human pathogen commonly found in atopic skin lesions, mediated food allergy sensitization may reveal novel therapeutic induces itch by directly acting on sensory neurons via V8 protease targets and provide insight into the skin-to-gut axis in food allergy (sspA), exacerbating skin damage. We hypothesized that S. aureus development. 142 Program for Research in Science and Engineering Reprogramming of Granulosa Cells via BTP and L1L2 Gene Disruption to Investigate Their Tumorigenic Potential in High-Grade Serous Ovarian Cancer Devin Morales, Jason Yu, Sam Van OOijen, Doeke Hekstra Harvard College | Quincy House | Chemical & Physical Biology and Applied Math | 2028 Aneuploidy, the gain or loss of chromosomes, is a leading improving brightness and photostability for clearer visualization of cause of genetic disorders such as Down syndrome, cancer, and chromosomal behavior. In the final step, I will select for a third other developmental conditions. My project aims to improve plasmid containing the plexAbox promoter, lacO, centromere 3, the effectiveness of a bio-orthogonal mechanism for inducing andkanamycinresistance. Inducingtranscriptionatthecentromere aneuploidy in Saccharomyces cerevisiae, enabling us to study disrupts kinetochore attachment, causing missegregation during this condition using Raman spectroscopy. The auxotrophic yeast telophase as the mitotic spindles retract—resulting in aneuploid strain used allows for straightforward selection in downstream daughtercells. ThelacI-lacOsystemwillbeusedtovisualizethese experiments. events, with lacO positioned downstream of centromere 3 and lacI tagged with mNeonGreen. The first phase involved amplifying a plasmid containing mCherry and a kanamycin-resistance gene using forward and reverse The project is ongoing. Future plans include completing primers with homologous tails flanking the HTA2 sequence, the transformations, analyzing different strains using Raman enabling targeted insertion into the genome. We then performed spectroscopy—a method that measures the vibrational energy of a transformation on YPD plates containing kanamycin to select for molecules through light energy—under varying environmental integration. This transformation allows for visualization of the cellnditions, and applying the mechanism to different chromosomes cycle, as HTA2 expression levels vary with cell cycle progression. to study broader effects. Ultimately, this project seeks to refine an Next, I plan to introduce another plasmid containing a synthetic existingmethodofinducinganeuploidybyincreasingitsefficiency and enabling clearer, real-time visualization. By integrating transcription factor with a plexAbox promoter recognition site, an this system with Raman spectroscopy, we hope to offer a more estrogen receptor, and an activation domain. This construct also quantitative and detailed approach to studying the molecular and includes the lacI repressor, a functionalHIS gene, and GFP. I will thenperformaGibsonassemblytoreplaceGFPwithmNeonGreen, structural dynamics of aneuploidy.

Source:

Harvard / Harvard College | Dunster House | Neuroscience | 2028 / 2025

Topics:

cell, food, sensitization, ovarian, skin, cancer, allergy, protease, antigen, mechanism, hgsoc, transformation

Co-authors:

@briannacarbajal406 , @xingengzhao407 , @chengwang408