The poster session will take place on Tuesday, February 25th from 4:00 - 5:30 PM. The session will be split so that odd-numbered posters will present for the first 45 minutes, and even-numbered posters will present for the second 45 minutes, to allow presenters an opportunity to enjoy the poster session as well.
Poster boards will be 4’x8’, with two posters per side per board. Please make sure your posters are no larger than 4’x4’.
All Trainee (Undergrad, Grad Student and Postdoc) Poster presenters will be considered for a poster prize. Please review the poster judging criteria here.
#1) Microbial Colonization and Its Tumor-Modulating Effects in CRC Organoids
Abhinav Bhushan, Illinois Institute of Technology
Scientific Abstract:
Gut bacteria colonize the intestinal tract, and its quantity and composition change during disease. Sometimes, it is necessary to prevent the colonization of specific bacterial species, such as E. coli O157. In other cases, it may be desirable to promote the colonization, such as probiotics. Diseases or inflammation influence the growth and colonization of bacteria; however, this is poorly understood. This study aims to create an experimental platform to study bacterial colonization of colon organoids and how the bidirectional signaling between the bacteria and colon is affected by colorectal cancer.
We used healthy and tumor colon organoids isolated from APCf/f CDX2-Cre mice and E. coli bacteria. The organoids were cultured in Matrigel for several days; after the organoids became mature, bacteria were added for 72 hours with a wash every 24 hours. The colonization and influence on gene expression were analyzed via confocal microscopy and qPCR respectively.
Successful colonization of the organoids was observed and confirmed with confocal imaging, which revealed bacterial penetration of the Matrigel and colonization. We assessed the gut-host cell interactions using specific markers and found bacteria to upregulate cell function markers, signifying that the organoids' stemness enhanced upon colonization.
Lay Abstract:
Gut bacteria colonize the intestinal tract, and its quantity and composition change during disease. Sometimes, it is necessary to prevent the colonization of specific bacterial species, such as E. coli O157. In other cases, it may be desirable to promote the colonization, such as probiotics. Diseases or inflammation influence the growth and colonization of bacteria; however, this is poorly understood. This study aims to create an experimental platform to study bacterial colonization of colon organoids and how the bidirectional signaling between the bacteria and colon is affected by colorectal cancer.
We used healthy and tumor colon organoids isolated from APCf/f CDX2-Cre mice and E. coli bacteria. The organoids were cultured in Matrigel for several days; after the organoids became mature, bacteria were added for 72 hours with a wash every 24 hours. The colonization and influence on gene expression were analyzed via confocal microscopy and qPCR respectively.
Successful colonization of the organoids was observed and confirmed with confocal imaging, which revealed bacterial penetration of the Matrigel and colonization. We assessed the gut-host cell interactions using specific markers and found bacteria to upregulate cell function markers, signifying that the organoids' stemness enhanced upon colonization.
Other Authors: Ishita Dasgupta, Yixiao Ma, Durga Prasad, Rangineni, Brennan Shapiro
#2) Differential Effects of Fluid Shear Stress on Macrophage Functionality and Polarization Introduction
Natalie Curry, Rice University
Scientific Abstract:
Macrophages encompass up to 50% of tumor mass and increased presence correlates with poor patient prognosis. Most macrophages at the site of the tumor have an M2 phenotype, these macrophages help create an immunosuppressive environment and promote tissue remodeling, however M1 macrophages are known to be pro-inflammatory and exhibit anti-tumoral functions. We propose that exposure of macrophages to physiological forces, such as fluid shear stress (FSS) that tumor cells experience during metastasis can influence the differentiation and polarization of macrophages during this process. Understanding this dynamic effect on macrophage polarization states will shed light regarding how tumor associated macrophages function as they enter the bloodstream during metastasis. Following exposure to constant low-intensity FSS for 1 hour using a cone-and-plate viscometer, M0, M1, and M2 macrophages showed increased pro-inflammatory transcription factors, where monocytes and M2 macrophages exposed to FSS exhibited enhanced phagocytic activity. This indicates that FSS could be affecting each macrophage subtype within circulating tumor clusters in ways that confer anti- and pro- tumor advantages in metastatic cancer. Interestingly, FSS affects unpolarized macrophages by increasing the secretion of pro-inflammatory cytokine TNF-⍺ but not IFN- γ and does not affect phagocytic activity. This suggests that shear stress helps to polarize M0 macrophages to a M1-like phenotype rather than an M2-like phenotype. Ongoing research is addressing the effects of macrophage polarization as it relates to cancer metastasis by co-culturing macrophages and cancer cells to understand the effects macrophages have on cancer cell survival.
Lay Abstract:
Immune cells play vital roles creating the tumor microenvironment, and thus can influence cancer progression and effectiveness of treatment modalities. Macrophages are one of the most plastic immune cell types, where they can switch what roles they preform depending on the present environment. Macrophages can make up a significant portion of solid tumors, where the role of macrophages present in the tumors tend to promote cancer cell survival and prevent other immune cells to infiltrate the tumors and lead to poor patient prognosis. As cancer begins to metastasize, cancer cells might form clusters with macrophages and be exposed to fluid shear stress (FSS) within the bloodstream. We propose that FSS will have a varying effect on different subpopulations of macrophages and could provide vital information about potential survivability of cancer cells clustered with macrophages and the likelihood of these clusters forming metastatic nodes. Because metastatic cancer has been proven to be more deadly across cancer types, it is important to understand and have more robust models for how cancer spreads. Having a better understanding of how immune cells impact metastatic cancers can lead to improved studies on effective and personalized therapies to target metastatic cancer.
Other Authors: Michael R. King Ph.D, Nichole S. Sarna, Abigail R. Fabiano
#3) Multiplex, high-throughput method to study cancer and immune cell mechanotransduction
Abigail Fabiano, Vanderbilt University
Scientific Abstract:
Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, the process when cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for direct and seamless flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced activation of the mechanosensitive ion channel (MSC) Piezo1 by examining the mechanism of apoptosis. These results were confirmed through employing GsMTx-4, an inhibitor of cationic MSCs. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm^2) increases activation of murine bone marrow-derived dendritic cells, providing valuable insights into potential new approaches for cancer treatments. Ongoing studies are examining the response of cultured cell aggregates that resemble clinical CTC samples and the way in which FSS enhances macrophage differentiation. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.
Lay Abstract:
Cancer metastasis, or the spread of cancer, is the leading cause of cancer-related deaths worldwide. One of the main physiological forces influencing cancer metastasis is fluid shear stress (FSS) in a patient’s bloodstream within the circulatory system. To better understand this phenomenon in the laboratory, we have developed novel, cost-effective protocols to advance and provide alternate multiplex, high-throughput methods tailored to be used with the commercially available Viaflo96 device. This allows researchers to study the effects of FSS on metastasis. These protocols offer scientists worldwide a powerful tool to investigate how the intrinsic forces of the circulatory system can be harnessed to target cancer cells and activate immune cells to provide therapeutic approaches. For instance, continuing and advancing studies of how we can enhance the response and activation of immune cells to forces in the bloodstream is imperative in cancer since tumor cells can evade immune surveillance. These methodologies may also easily be adapted and modified based on a laboratory’s research needs and can be integrated with an unlimited array of downstream analysis methods. This research is significant because we provide further evidence regarding how we can develop innovative cancer therapeutics on a larger scale, providing a more personalized therapy for patients.
Other Authors: Spencer C. Robbins, Samantha V. Knoblauch, Schyler J. Rowland, Jenna A. Dombroski, Michael R. King
#4) CXCL7 Enhances Colon Cancer Cell Proliferation, Cell Viability, and Glucose Utilization
Michael Greene, Auburn University
Scientific Abstract:
Colorectal cancer ranks among the top three prevalent cancers worldwide. Chemokine (C-X-C motif) ligand 7 (CXCL7) – a protein product of the pro platelet basic protein (PPBP) gene – is a potential biomarker for colorectal cancer (CRC) diagnosis. In this study, we examined the role of CXCL7 in colon cancer cell proliferation through enhanced aerobic glycolysis and cell viability.
The effect of CXCL7 on cellular proliferation and aerobic glycolysis was examined using: 1) human colon cancer (HT-29) cells transfected with CXCL7 or an empty vector; 2) HT-29 cells treated with CXCL7 or control medium; and 3) CL-40 colon cancer CRISP-Cas9 knockdown (KD) cells with a partial CXCL7 KD or full CXCL7 KD. In addition, cell proliferation, cell viability and cancer colony growth were examined using 3D engineered tissues created from HT-29 overexpressing CXCL7 or vector and CL-40 CXCL7 KD or control lines.
We observed increased proliferation rate and lactate secretion into the media in HT-29 cells overexpressing or stimulated with CXCL7 and then reduced in CXCL7 KD CL-40 cell lines. Similar findings were observed in 3D engineered tissues created from HT-29 overexpressing CXCL7 or vector and CL-40 CXCL7 KD or control lines. In the 3D engineered tissues, cell viability was increased in tissues created from HT-29 overexpressing CXCL7 but decreased in CL-40 CXCL7 KD tissue. Glucose uptake and glycolytic flux assays confirmed the ability of CXCL7 to stimulate aerobic glycolysis.
Our study for the first time showed that CXCL7 stimulates colon cancer cell proliferation, enhances aerobic glycolysis, and increases cell viability.
Lay Abstract:
Colorectal cancer ranks among the top three prevalent cancers worldwide. Chemokine (C-X-C motif) ligand 7 (CXCL7) – a protein product of the pro platelet basic protein (PPBP) gene – is a potential biomarker for colorectal cancer (CRC) diagnosis. In this study, we examined the role of CXCL7 in colon cancer cell growth through enhanced use of glucose and the ability of cells to remain alive.
The effect of CXCL7 on cell growth and use of glucose was examined using: 1) human colon cancer that overexpress the CXCL7 protein; 2) HT-29 cells treated with CXCL7 protein; and 3) another human colon cancer cell line (CL-40) in which the expressing of CXCL7 was partially or fully blocked. In addition, 3D engineered tissues created from CXCL7 overexpressing or CL-40 reduced expressing cells was tested.
We observed increased cell growth and use of glucose in HT-29 cells stimulated with CXCL7 and then reduced in CXCL7 low expressing cell lines. Similar findings were observed in 3D engineered tissues. In the 3D engineered tissues, the ability of cells to remain alive was increased in tissues created from cells overexpressing CXCL7 but decreased in CL-40 reduced expressing cells.
Our study for the first time showed that CXCL7 stimulates colon cancer cell growth and enhances use of glucose and the ability of cells to remain alive.
Other Authors: Hadeel Aldhowayan, Jannatul Ferdous Nipa, Kathryn Edmondson, Ifeoluwa Odeniyi, Elizabeth A. Lipke
#5) Investigating the Effects of Tensile Strain on Breast Cancer Cell Dormancy Using a Lung-Mimetic Magnetic Actuation Platform
Madison Howard, Purdue University
Scientific Abstract:
Breast cancer (BC) often metastasizes to organs experiencing high mechanical stress, including the lungs. Despite this knowledge, the effect that dynamic forces native to the lungs have on early disseminated tumor cells is a currently under explored area of the metastatic cascade. Recent in vitro findings suggest BC cells enter a dormant state in response to tensile strain, yet the mechanisms by which these cells adapt to the dynamic conditions at metastatic sites remain unclear. In this work, we used a lung-mimetic magnetic actuation cell culture platform to apply tensile strain at various amplitudes and frequencies to MDA-MB-231 BC cells. The platform utilizes a fibrillar fibronectin (FN) matrix as the cell seeding substrate, a key component of the early metastatic niche, making it a more physiologically relevant microenvironment. In this study, changes in cellular morphology, proliferation and gene expression between cyclic stretching, constant stretching and static conditions were evaluated. We found applying tensile stretch decreased overall cell metabolic activity but did not affect cell viability, indicating the cells entered a dormant state. Applying tensile stretch to the FN substrate changed the cell morphology to be consistent with previously reported senescent cells. Further, RNA sequencing of the cells after four days of stretching identified differentially expressed genes compared to the static control group. This effect was amplified in the constant stretch group, where additional DEGs were identified. Additionally, we utilized fluorescent reporter cells to compare DNA damage accumulation between the different loading conditions. Based on our RNA sequencing results, which showed an increase in Insulin-like growth factor-binding protein 3 (IGFBP3), we hypothesize DNA damage accumulation could be causing this dormant state in response to tensile stretch. These findings seek to elucidate mechanisms of BC dormancy and mechanical adaptation, offering new insights into how metastatic cells survive within the dynamic conditions of the lung microenvironment.
Lay Abstract:
Breast cancer (BC) often spreads to the lungs, which are exposed to constant mechanical forces during respiration. However, we don’t fully understand how these forces affect the cancer cells that have spread there. Recent work from our group suggests that stretching may push BC cells into a dormant state, but the underlying mechanisms are unclear. In this study, we used a magnetic actuation system to mimic the lung environment to apply different amounts and frequencies of stretching forces to MDA-MB-231 BC cells. The cells were grown on a fibronectin (FN) matrix, which resembles the environment these cells encounter in the body. We studied how these forces affected cell morphology, proliferation, and gene expression. We found that stretching decreased the cells' overall metabolic activity but didn’t kill them, suggesting they entered a dormant state. The stretched cells also changed shape similar to dormant cells. When we analyzed their gene activity, we found specific genes, including IGFBP3, were upregulated, especially under constant stretching. We believe this gene may play a role in dormancy through a DNA damage repair mechanism. These results shed light on how BC cells survive and adapt to the lung’s dynamic environment by becoming dormant, offering potential new directions for treating metastatic cancer.
Other Authors: Luis Solorio
#6) Ectopic ATP Synthase as a Novel Dependency for Hypoxic Adaptation in Retinoblastoma
Samantha McLaughlin, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine
Scientific Abstract:
Hypoxia is a common phenomenon in advanced tumors, yet the effect of hypoxia on metabolic reprogramming remains unclear. In retinoblastoma (RB), we have shown that estrogen-related receptor gamma (ESRRG) undergoes continuous positive selection during tumor evolution and is upregulated by hypoxia. Transcriptional analysis revealed that ESRRG is pivotal in mediating metabolic reprogramming, with cells displaying preferential aerobic fermentation and microenvironment acidification. Here, we show that hypoxic stress and ESRRG deregulation leads to increased ectopic expression of ATP synthase on the plasma membrane, where it is critical to cancer cell proliferation. Translocation of ATP synthase to the plasma membrane was increased in a time-dependent manner when RB cells were exposed to hypoxia. Both the F0 and F1 domains were identified on the outer plasma membrane by confocal microscopy. Intracellular ATP concentration, measured by luciferase reaction, was significantly increased by media acidification. Inhibition of lactate/proton export through monocarboxylate transporters resulted in media alkalinization and a significant decrease in intracellular ATP, which could be rescued by exogenous acidification of the media, suggesting that ectopic ATP synthase uses the proton motive force generated by extracellular acidification to synthesize ATP. A novel F0-targeting ATP synthase inhibitor resulted in near complete cell death of RB cells but showed minimal effect on non-malignant control cells. These findings suggest that ATP synthase on the RB cell surface plays an important role in metabolic adaptation and cell proliferation. These results demonstrate that ATP synthase inhibitors targeting the F0 domain may represent a new strategy to target cancer cells.
Lay Abstract:
Cancer cells adapt to low oxygen levels (hypoxia) by changing how they produce energy, but the exact details of this process are not fully understood. In retinoblastoma (RB), a pediatric intraocular tumor, we have shown that estrogen-related receptor gamma (ESRRG) becomes increasingly active as the cancer develops and plays a key role in changing how cancer cells generate energy. Specifically, the combination of ESRRG upregulation and hypoxia makes RB cells more reliant on aerobic fermentation, which acidifies their surroundings. Here, we found that under hypoxia, RB cells move ATP synthase from inside the cell to the surface, where it uses the acidic environment outside of the cell to produce energy in the form of intracellular ATP. Blocking the cells’ ability to export acid disrupted this process, reducing energy levels in the cells. Interestingly, we found that a new drug targeting the F0 domain of ATP synthase killed nearly all RB cells without affecting healthy cells. This suggests the drug specifically disrupts energy production at the cell surface, which is unique to cancer cells. Our findings highlight how cancer cells adapt to harsh conditions like low oxygen by rewiring their energy production. Targeting this unique energy-making process could lead to new, more precise cancer treatments.
Other Authors: Zelia Correa, Daniel Pelaez
#7) Fluid Assisted Transformation and Dissemination of Fallopian Tube Epithelial Cells
Geeta Mehta, University of Michigan
Scientific Abstract:
High grade serous ovarian carcinoma (HGSC) is the most common type and most lethal gynecologic cancer due to its late-stage diagnosis. One of the cells of origin of HGSC reside in the fimbria of the fallopian tube. The cells lining the lumen of the fallopian tube are stimulated with varying levels of shear stress, which can vary in magnitude in the different phases of the menstrual cycle, pre- and post-menopause under normal healthy conditions. The role of shear stress in the fallopian tubes is well recognized for the transport of gametes and embryo. However, the role of fluid flow and shear stress in the fallopian tube epithelium remains unexplored in the context of premalignant fallopian tube epithelial secretory cells (FTSEC), that may undergo early precursor escape and result in HGSC initiation. Our central hypothesis is that shear stress stimulation transforms the FTSEC in the fimbria to activate EMT pathways and results in their dissemination in the peritoneum. We are working towards the following Aims to address the scientific knowledge gap and the central hypothesis: 1) Characterize fluid assisted transformation and dissemination in FTSEC within custom-built microphysiological systems. 2) Determine the bulk viscoelastic and mechanical properties of the healthy human fallopian tubes and their local heterogeneity. 3) Identify and validate the mechanotransduction pathway governing the transformation of FTSEC under shear stress stimulation. By completing this work, we expect to develop new knowledge about molecular mechanisms involved in the mechanobiology of shear stress driven programming of FTSEC.
Lay Abstract:
Many high-grade serous ovarian cancers originate from the fallopian tubes, from where the shedding of transformed fallopian tube secretory cells leads to metastasis. In this work, we will investigate if the fluidic environment around transformed fallopian tube cells has any roles in their release within the peritoneal organs. With the in vitro and in vivo models developed in our labs, we will establish the importance of shear stresses in the fluidic niches of fallopian tubes, that could be utilized in early detection of ovarian cancers.
Other Authors: Raneem Ahmad, Isha Bhorkar, Nina Treacher, Eric Horst, Analisa DiFeo, Mike Solomon, Ron Larson
#8) Collagen Architecture Modulates Immune-Mediated Killing of Breast Tumor Cells
Faith Muriuki, Cornell University
Scientific Abstract:
Introduction: Cell-based immunotherapy relies on collecting and manipulating immune cells to enhance their ability to recognize and negate the attempts of cancer cells to bypass or evade an immune response [1]. They have shown great promise in treating and curing various types of blood cancers. For example, T-cell therapy has successfully treated lymphoma [2]. However, the success in treating blood cancers has not translated to solid tumors. Clinical trials targeting solid tumors have achieved limited efficacy in tumor treatment [2]. Recent studies have demonstrated that mechanical forces have a significant influence on the ability of immune cells to eliminate tumor cells [3-4]. To initiate the release of cytotoxins, immune cells must first establish an immune synapse, which serves as the connection between immune and target cells [3]. At the molecular level, the receptors involved in forming the immune synapse have been shown to be regulated by mechanical forces [4]. At the cellular level, immune cells exert tension on the tumor cell to facilitate the formation of the immune synapse and transmission of cytotoxins [3]. While this pioneering research highlights the importance of mechanical forces in immune killing, it is essential to note that these experiments were conducted on a 2D substrate. However, solid tumors are native to 3D environments, where cellular behavior differs significantly from those in 2D environments [5]. Thus, illustrating the importance of conducting experiments that investigate the impact of mechanical forces on immune killing in 3D platforms. We hypothesize that mechanical forces can be manipulated to enhance immune killing of solid tumors.
Materials and Methods: I used a 3D collagen matrix made from type I collagen to better mimic the physiologically realistic breast tumor microenvironment [6]. The collagen concentrations were 1.5 mg/mL and 3.5 mg/mL, resulting in a tissue stiffness range of 6-900 Pa. This range falls within the spectrum of normal (60 Pa) to malignant (1300 Pa) breast tissue stiffness [7]. Additionally, I utilized natural killer (NK) cells, specifically, NK-92 MI cells [8], which are transfected with IL-2 cDNA. This modification allows them to remain active without the need for external stimulation. Both MCF-7 cells and NK cells were embedded into 3D collagen matrices of 1.5 mg/mL and 3.5 mg/mL collagen. To distinguish between the two cell lines, the MCF-7 cells were labeled with a GFP reporter. Images were captured every 10 minutes for 22 hours using an epi-fluorescence microscope. NK cell-mediated killing was indicated by the loss of MCF-7 fluorescence. MCF-7 fluorescence was quantified over the 22-hour period.
Results and future perspectives: My experimental results revealed that the combination of MCF-7 cells and NK cells in 1.5 mg/mL collagen matrix resulted in the highest MCF-7 cell deaths, in comparison to those in 3.5 mg/mL collagen gel. The results indicate that a 1.5 mg/mL collagen concentration provides the most favorable environment for NK cell-mediated killing of MCF-7 cells. This suggests that pore size influences immune-cancer cell interactions. Previous studies have shown that the pore size of a 3.5 mg/mL collagen matrix is less than 1 µm, which is smaller than the size of a cell nucleus [9]. A smaller pore size likely results in greater restriction of NK cell motility. Future work will further investigate the effect of pore size on NK motility through glycation of collagen matrices.
Acknowledgments: This work is supported by R01CA221346-03. I thank Lance R. Collins Fellowship from Cornell University's College of Engineering for the support. I also want to thank my lab members: Brian Cheung, Cass Nordmann, Mrinal Pandey and Young Joon Suh.
References: [1] Boyiadzis, M.M.,et. al. Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance. J Immunother Cancer. (2018). [2] Wang M et al. Current advances in T-cell-based cancer immunotherapy. Immunotherapy. (2014).[3] Jiang, H.D et al., Molecular Tug of War Reveals Adaptive Potential of an Immune Cell Repertoire. Phy Rev X. (2023). [4] Wang, M.S., et al. Mechanically active integrins target lytic secretion at the immune synapse to facilitate cellular cytotoxicity. Nat Commun (2022). [5] Basu, R., et al. Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing. Cell. (2016). [6] Cukierman, E. et al. Taking cell-matrix adhesions to the third dimension. Science. (2001). [7] Paszek, M.J., et al. Tensional homeostasis and the malignant phenotype. Cancer Cell. (2005). [8] ] Zhao, X., et al. Cord-Blood Natural Killer Cell-Based Immunotherapy for Cancer. Front Immunol. (2020). [9] Hall, M.S., et al., Fibrous nonlinear elasticity enables positive mechanical feedback between cells and ECMs. Proc Natl Acad Sci U S A. (2016).
Lay Abstract:
Introduction: Cell-based immunotherapy trains the body’s immune system to better fight cancer [1]. It has shown great promise in treating lymphoma and other single-cell blood cancers [2]. However, this success has not translated to solid tumors, as clinical trials targeting these cancers have achieved only limited results [2]. Recent studies have revealed that mechanical forces significantly influence how immune cells kill tumor cells [3–4]. To eliminate tumor cells, immune cells must first establish a region of physical contact with the tumor cell, known as an “immune synapse” [3]. This process is shaped by mechanical forces at both the cellular level—where immune cells apply physical tension on the tumor cell [3]—and the molecular level, as the receptors involved are regulated by mechanical forces [4]. It is important to note that these groundbreaking experiments were conducted on flat, 2D surfaces. Since solid tumors exist in 3D environments, and cells behave differently in 2D versus 3D settings [5], it is crucial to study how mechanical forces affect immune cells under conditions that more closely resemble real tumors. We hypothesize that a deeper understanding of mechanical forces will allow us to manipulate them to enhance the ability of immune cells to kill solid tumors.
Materials and Methods: I used a 3D collagen platform to more accurately replicate the natural breast tumor microenvironment (TME). Collagen was used because it is both the most abundant structural protein in the body and is a key component of the TME [6]. The collagen was prepared at two different concentrations (1.5 mg/mL and 3.5 mg/mL), resulting in stiffness levels ranging from soft (6 Pa) to much stiffer (900 Pa). These levels span the typical stiffness of healthy breast tissue (around 60 Pa) and approach the stiffness of cancerous breast tissue (approximately 1300 Pa) [7]. For the experiment, I used a type of immune cell called natural killer (NK) cells, specifically NK-92 MI cells [8], which were genetically modified to remain active without external stimulation. Both cancer cells (MCF-7) and NK cells were embedded into the 3D collagen platforms at the two collagen concentrations. To differentiate between the two cell types, the cancer cells were labeled with a green fluorescent protein (GFP), which causes them to glow under a microscope. Images were taken every 10 minutes over a 22-hour period using an epi-fluorescence microscope. When an NK cell successfully killed a cancer cell, the green glow from the cancer cell disappeared, marking its death. By measuring the decrease in green fluorescence over time, I was able to monitor how effectively the NK cells killed the cancer cells.
Results and future perspectives: My experimental results showed that the highest cancer cell death occurred when MCF-7 cancer cells and NK cells were placed in the 1.5 mg/mL collagen platform, compared to the stiffer 3.5 mg/mL collagen platform. These findings suggest that the 1.5 mg/mL collagen concentration creates a more favorable environment for NK cells to effectively kill MCF-7 cancer cells. This result points to the possibility that the size of the gaps (pores) within the collagen matrix plays a key role in how well immune cells interact with cancer cells. Previous research has shown that the pore size of a 3.5 mg/mL collagen matrix is particularly small, less than 1 µm, which is smaller than the size of a cell nucleus [9]. Such small pores likely restrict the movement of NK cells, making it harder for them to travel through the collagen and efficiently reach the cancer cells in order to be able to kill them. Future research will explore the effect of pore size on NK cell movement by using modified collagen platforms.
Acknowledgments: This work is supported by R01CA221346-03. I thank Lance R. Collins Fellowship from Cornell University's College of Engineering for the support. I also want to thank my lab members: Brian Cheung, Cass Nordmann, Mrinal Pandey and Young Joon Suh.
References: [1] Boyiadzis, M.M.,et. al. Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance. J Immunother Cancer. (2018). [2] Wang M et al. Current advances in T-cell-based cancer immunotherapy. Immunotherapy. (2014).[3] Jiang, H.D et al., Molecular Tug of War Reveals Adaptive Potential of an Immune Cell Repertoire. Phy Rev X. (2023). [4] Wang, M.S., et al. Mechanically active integrins target lytic secretion at the immune synapse to facilitate cellular cytotoxicity. Nat Commun (2022). [5] Basu, R., et al. Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing. Cell. (2016). [6] Cukierman, E. et al. Taking cell-matrix adhesions to the third dimension. Science. (2001). [7] Paszek, M.J., et al. Tensional homeostasis and the malignant phenotype. Cancer Cell. (2005). [8] ] Zhao, X., et al. Cord-Blood Natural Killer Cell-Based Immunotherapy for Cancer. Front Immunol. (2020). [9] Hall, M.S., et al., Fibrous nonlinear elasticity enables positive mechanical feedback between cells and ECMs. Proc Natl Acad Sci U S A. (2016).
Other Authors: Brian Cheung, Mrinal Pandey, Jeffrey E. Segall, and Mingming Wu
#9) Role of ovulatory factors on ovarian tumorigenesis from the fallopian tube epithelium
Angela Russo, University of Illinois at Chicago
Scientific Abstract:
High-Grade serous ovarian cancer (HGSOC) is the most prevalent and lethal form of ovarian cancer, and the severity of the prognosis is attributed to the absence of early detection methods that leads to late-stage diagnoses. Emerging evidence points to early preneoplastic lesions primarily manifesting in the fallopian tube epithelium (FTE), implicating it as the main origin of HGSOC. Despite this insight, the molecular mechanisms driving the early development of this disease remain elusive.
Notably, ovulation has been identified as a risk factor for ovarian cancer. In fact, measures such as oral contraceptive, salpingectomy, and pregnancies, which impede ovulation, have demonstrated a reduction in ovarian cancer risk. Our research aims to delineate the ovulation-mediated changes in the fallopian tube epithelium that may initiate early tumorigenesis. To achieve this, we employed the PREDICT-MOS microfluidic system, recreating distinct phases of the menstrual cycle on this dynamic flow platform. Ovulatory secretions were isolated and applied to human fallopian tube tissues ex vivo. Strikingly, spatial transcriptomic unveiled an upregulation of genes involved in glucose metabolism and oxidative stress in response to ovulatory secretion. Expanding this analysis, mass spectrometry was employed to identify proteins secreted pre- and post-ovulation. The findings underscored that ovulation enriches the secretion of proteins involved in oxidative stress regulation and glycolysis including TXNIP. These results collectively shed light on the intricate molecular dynamics triggered by ovulation in the fallopian tube epithelium, potentially contributing to early tumorigenic events. This research holds promise in unraveling critical pathways for the development of targets preventive and therapeutic strategies for HGSOC.
Lay Abstract: High-Grade serous ovarian cancer (HGSOC) is the most prevalent and lethal form of ovarian cancer, and the severity of the prognosis is attributed to the absence of early detection methods that leads to late-stage diagnoses. Emerging evidence points to early preneoplastic lesions primarily manifesting in the fallopian tube epithelium (FTE), implicating it as the main origin of HGSOC. Despite this insight, the molecular mechanisms driving the early development of this disease remain elusive.
Notably, ovulation has been identified as a risk factor for ovarian cancer. In fact, measures such as oral contraceptive, salpingectomy, and pregnancies, which impede ovulation, have demonstrated a reduction in ovarian cancer risk. Our research aims to delineate the ovulation-mediated changes in the fallopian tube epithelium that may initiate early tumorigenesis. To achieve this, we employed the PREDICT-MOS microfluidic system, recreating distinct phases of the menstrual cycle on this dynamic flow platform. Ovulatory secretions were isolated and applied to human fallopian tube tissues ex vivo. Strikingly, spatial transcriptomic unveiled an upregulation of genes involved in glucose metabolism and oxidative stress in response to ovulatory secretion. Expanding this analysis, mass spectrometry was employed to identify proteins secreted pre- and post-ovulation. The findings underscored that ovulation enriches the secretion of proteins involved in oxidative stress regulation and glycolysis including TXNIP. These results collectively shed light on the intricate molecular dynamics triggered by ovulation in the fallopian tube epithelium, potentially contributing to early tumorigenic events. This research holds promise in unraveling critical pathways for the development of targets preventive and therapeutic strategies for HGSOC.
Other Authors: Angela Russo
#10) A 3D bioprinted immunocompetent and perfusable model of neuroblastoma to study the tumor microenvironment impact on therapy response
Mehdi Salar Amoli, Emory University and Georgia Institute of Technology
Scientific Abstract:
Introduction: The neuroblastoma (NB) tumor microenvironment (TME) contains immune cells, fibroblasts (CAFs), endothelial cells, extracellular matrix and vascular flow that impact therapy response. Current in vitro models fail to account for all TME components and humanized mouse models are low throughput. This study aims to integrate 3D bioprinting, NB spheroids, immune cells and perfusion bioreactor technologies to establish a new generation of tunable, vascular analogues for investigating NB TME contributions to therapy resistance.
Methods: Embedded extrusion bioprinting with gelatin methacryloyl (GelMA) was used to develop a model with vascular channels surrounding a central cavity. Human umbilical vein endothelial cells (HUVECs) were seeded into the vascular channels. Human-derived NB spheroids were introduced into the central cavity. Peripheral blood mononuclear cells (PBMCs) were injected into the constructs. Constructs were cultured under static and perfusion dynamic conditions. Brightfield microscopy, live/dead imaging, flow cytometry and immunohistochemistry (IHC) were used to analyze cellular components.
Results: We optimized a media that sustains viability of HUVECs, NB spheroids, and PBMCs for up to 10 days of co-culture. HUVECs formed a uniform endothelial layer and migrated into the NB spheroid under dynamic conditions. IHC of NB spheroids under perfusion showed features of increased growth/migration. IHC and flow cytometry demonstrated ratios of PBMCs within NB spheroids similar to primary tumors.
Conclusions: Perfused immunocompetent 3D bioprinted NB models sustain cellular viability and phenotype, providing a high throughput model for therapy testing. Vascular flow promotes cellular interactions. Gene expression of all cells and primary tumor validation are ongoing.
Lay Abstract:
Despite advancement of multimodal therapies incorporating strategies such as chemotherapy and immunotherapy, high risk neuroblastoma still has a mortality rate exceeding 50 %. A significant challenge facing the treatment strategies is the resistance of tumors within their natural environment to therapies that have shown efficacy in laboratory settings. This resistance is thought to be partially attributed to the highly complex tumor microenvironment (TME), which is composed of a variety of cell types, as well as a complex extracellular matrix (ECM). Properties of this ECM, such as its stiffness, are known to have a major impact on success of treatment strategies. However, lack of robust in vitro models or animal models capable of replicating this complex TME with high precision has resulted in a limited ability to analyze the effect of different parameters causing resistance to therapy. Recent tissue engineering strategies, such as 3D bioprinting and perfusion bioreactors, have created optimism in recapitulating this complex TME in a laboratory setup enabling further studies on the root causes of therapy resistance in neuroblastoma. Consequently, this study aims to develop a 3D bioprinted in vitro model of neuroblastoma incorporating the tumor microtissues in the form of spheroids, vasculature, and different cell types present in the TME. Consequently, this study will be able to generate a deeper understanding of the mechanisms of tumor progression and its response to therapy, and serve as a research enabling platform for identification of more effective clinical options.
Other Authors: Jenny Shim, Hunter Jonus, Krista Barbour Alexande, Morgan McCraw, Martin Tomov, Sarah Rezapourdamanab, Yamini Sing, Emmah Howard, Margaret Wade, Adithya Jyothish Pillai, Kelly Goldsmith, and Vahid Serpooshan
#11) Organ-on-Chip Model of Colorectal Cancer Demonstrates PIEZO1 Driven Tumor Aggressiveness
Curran Shah, University of Southern California
Scientific Abstract:
While mechanical forces have long been known to affect the progression of cancer, until recently traditional model systems were either too simple or complex to accurately study these forces. With the advent of organ-on-chip (OOC) technology, models can now integrate human-relevant physiological forces in a setting conducive for their study. In this study, we explore the influence of peristaltic forces on the early metastatic spread of colorectal cancer. Specifically, we investigate how these mechanical forces enhance the invasive capability of colon epithelial cells. Our microfluidic OOC combines an epithelial channel together with an endothelial channel via a porous membrane allowing for a simulation of early metastatic spread into the vasculature. Vacuum channels along the tube length allow for the application of forces in a cyclic manner to mimic peristaltic forces. Analysis of gene and protein expression found that peristaltic forces caused changes in the phosphorylation levels of the transcription factor YAP as well as changes in epithelial-to-mesenchymal (EMT) pathways resulting in greater invasion of tumor cells from the epithelial channel into the endothelial channel shown by on-chip confocal imaging. Knockdown studies demonstrated that the response to peristaltic forces is driven through a PIEZO1-mediated mechanism. Our findings demonstrate that the mechanical forces of the tumor microenvironment can dramatically alter the tumor behavior.
Lay Abstract:
Mechanical forces (muscle contractions, fluid pressure, etc.) have long been known to dramatically alter the progression of cancer, but traditional cell culture and animal models have proven inadequate for their study. Often these models are either too simple to include physiologically relevant forces or too complex to effectively isolate and study these subtle forces. Organ-on-chip (OOC) technology offers the ability to address these issues by allowing for the combination of human cells and physiologically relevant forces in a manner that still allows for their accurate study. Using this technology, we developed a model of colorectal cancer that incorporates colon tumor cells, vascular cells, and peristaltic forces (the natural motions responsible for gut motility) in order to study how peristaltic forces affected the ability of colon tumor cells to invade and spread into the vasculature. Our findings demonstrated the ability of peristaltic forces to enhance the number of early metastatic invasion events. Additionally, we found that peristaltic forces induced biological changes in both gene and protein expression of the tumor cells to enhance their invasive capability. Subsequently, we investigated the proteins responsible for responding to these peristaltic forces studies and found that the response was mediated by the PIEZO1 protein. In summary, we have developed an OOC model of colorectal cancer that incorporated peristaltic forces for more accurate study and found that these forces could dramatically impact the invasive capability of the cancer cells via the PIEZO1 protein.
Other Authors: Carly Strelez, Aaron Schatz, Hannah Jiang, Rachel Perez, Shannon M. Mumenthaler