{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/140686"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/140686","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Engineering Tumor-Targeting Bacteria and Characterizing Their Interactions with Tumor Cells in Therapy-Resistant Breast Cancers","abstract":"Breast cancer (BC) accounts for one-third of malignancies among women in 157 countries and ~15% mortality among diagnosed cases, a burden projected to reach 1.1 million deaths per year by 2050. Five-year survival drops from >90% in early-stage BC to ~32% for therapy-resistant subtypes such as triple-negative (TNBC), hormone-receptor–variable or resistant ER+, and Luminal B tumors. For these high-risk subtypes, molecular heterogeneity undermines targeted therapies, and clinical management still relies on maximum tolerable doses of systemic chemotherapy, causing severe dose-limiting toxicities. Moreover, the dense collagen-rich extracellular matrix (ECM) of solid tumors restricts intratumoral drug transport, motivating strategies that function across BC subtypes, overcome ECM barriers, and inform lowered clinical dosing. Bacteria-based cancer therapy (BBCT) with cancer-selective bacteria combines motility, self-replication, and on-board biosynthesis with the programmability of synthetic biology, enabling local release of therapeutic factors within the tumor microenvironment. Attenuated Salmonella Typhimurium VNP20009 (ST) exhibits ~10³-10⁴-fold tumor selectivity with respect to liver and spleen. It has a favorable clinical safety profile but remains inefficacious due to poor colonization. Our lab previously showed that ECM-targeting ST with collagenase secretion improved tumor penetration without gross collagen disruption, but at the cost of reduced bacterial fitness and motility. In this dissertation, we hypothesized that a fitness-restored, ECM-targeting ST enhances bacterial intratumoral transport and colonization, as well as chemotherapy penetration. We evaluated the engineered strains in perfused 3D tumor models that represent in vivo intratumor transport properties and investigated cancer cells-neutrophil interactions in presence of bacterial factors. First, we developed a high-motility, fitness-improved collagenase-expressing strain (HM-CEST ΔydcP) that preserves 100% motility under sub-cytotoxic chemotherapy. This strain improves intratumoral transport, and reduces spheroid viability and tumor migration relative to chemotherapy alone while maintaining tumor specificity and safety in preclinical murine models in vivo. Second, we validated a perfusion-enabled microfluidic spheroid platform that supports at least 14-day culture of murine TNBC and ER+ spheroids, enabling long-term BBCT screening. We demonstrated that perfused spheroids require lower chemotherapy doses than static cultures. Third, we biophysically characterized the crosstalk between BBCT, neutrophils, and Luminal B BC cells, demonstrating that neutrophils in the presence of bacteria supernatant suppress cancer cell growth, viability, and migration. Collectively, this work delivers a fitness-restored ECM-targeting Salmonella chassis, validates a perfusion-enabled 3D tumor-spheroid microphysiological platform, and develops a quantitative framework for neutrophil–bacteria–cancer cell interactions, contributing to the design pipeline for next-generation BBCT and supporting the long-term goal of safer, more affordable, lower-dose, and more broadly applicable therapies for therapy-resistant breast cancers.","abstract_html":"Breast cancer (BC) accounts for one-third of malignancies among women in 157 countries and ~15% mortality among diagnosed cases, a burden projected to reach 1.1 million deaths per year by 2050. Five-year survival drops from >90% in early-stage BC to ~32% for therapy-resistant subtypes such as triple-negative (TNBC), hormone-receptor–variable or resistant ER+, and Luminal B tumors. For these high-risk subtypes, molecular heterogeneity undermines targeted therapies, and clinical management still relies on maximum tolerable doses of systemic chemotherapy, causing severe dose-limiting toxicities. Moreover, the dense collagen-rich extracellular matrix (ECM) of solid tumors restricts intratumoral drug transport, motivating strategies that function across BC subtypes, overcome ECM barriers, and inform lowered clinical dosing. Bacteria-based cancer therapy (BBCT) with cancer-selective bacteria combines motility, self-replication, and on-board biosynthesis with the programmability of synthetic biology, enabling local release of therapeutic factors within the tumor microenvironment. Attenuated Salmonella Typhimurium VNP20009 (ST) exhibits ~10³-10⁴-fold tumor selectivity with respect to liver and spleen. It has a favorable clinical safety profile but remains inefficacious due to poor colonization. Our lab previously showed that ECM-targeting ST with collagenase secretion improved tumor penetration without gross collagen disruption, but at the cost of reduced bacterial fitness and motility. In this dissertation, we hypothesized that a fitness-restored, ECM-targeting ST enhances bacterial intratumoral transport and colonization, as well as chemotherapy penetration. We evaluated the engineered strains in perfused 3D tumor models that represent in vivo intratumor transport properties and investigated cancer cells-neutrophil interactions in presence of bacterial factors. First, we developed a high-motility, fitness-improved collagenase-expressing strain (HM-CEST ΔydcP) that preserves 100% motility under sub-cytotoxic chemotherapy. This strain improves intratumoral transport, and reduces spheroid viability and tumor migration relative to chemotherapy alone while maintaining tumor specificity and safety in preclinical murine models in vivo. Second, we validated a perfusion-enabled microfluidic spheroid platform that supports at least 14-day culture of murine TNBC and ER+ spheroids, enabling long-term BBCT screening. We demonstrated that perfused spheroids require lower chemotherapy doses than static cultures. Third, we biophysically characterized the crosstalk between BBCT, neutrophils, and Luminal B BC cells, demonstrating that neutrophils in the presence of bacteria supernatant suppress cancer cell growth, viability, and migration. Collectively, this work delivers a fitness-restored ECM-targeting Salmonella chassis, validates a perfusion-enabled 3D tumor-spheroid microphysiological platform, and develops a quantitative framework for neutrophil–bacteria–cancer cell interactions, contributing to the design pipeline for next-generation BBCT and supporting the long-term goal of safer, more affordable, lower-dose, and more broadly applicable therapies for therapy-resistant breast cancers.","abstract_has_math":false,"creators":["Saha, Binita"],"institution":"Virginia Tech","degree_name":"Doctor of Philosophy","degree_level":"doctoral","degree_discipline":"Macromolecular Science and Engineering","degree_department":"Graduate School","school":null,"contributors":[],"advisors":[],"committee_chairs":["Behkam, Bahareh"],"committee_members":["Li, Liwu","Bortner, Michael J.","Johnson, Blake","Senger, Ryan S."],"year":2026,"date_issued":"2026-01-08","date_published":"2026-01-08","updated_at":"2026-07-22T22:18:55Z","subjects":["Breast cancer","microphysiological 3D cancer models","combinatorial therapy","bacteria-based cancer therapy","extra cellular matrix targeting","immunotherapy"],"languages":["en"],"rights":["Creative Commons Attribution 4.0 International"],"rights_urls":["http://creativecommons.org/licenses/by/4.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:45381"],"render_values":[{"text":"vt_gsexam:45381","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/10919/140686","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Behkam, Bahareh"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Li, Liwu","Bortner, Michael J.","Johnson, Blake","Senger, Ryan S."]},{"key":"dc:contributor.department","label":"Department","values":["Graduate School"]},{"key":"dc:creator","label":"Author","values":["Saha, Binita"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-01-09T09:00:20Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-01-09T09:00:20Z"]},{"key":"dc:date.issued","label":"Date","values":["2026-01-08"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Macromolecular Science and Engineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Breast cancer","microphysiological 3D cancer models","combinatorial therapy","bacteria-based cancer therapy","extra cellular matrix targeting","immunotherapy"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Creative Commons Attribution 4.0 International"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://creativecommons.org/licenses/by/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:45381"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10919/140686"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Breast cancer (BC) accounts for one-third of malignancies among women in 157 countries and ~15% mortality among diagnosed cases, a burden projected to reach 1.1 million deaths per year by 2050. Five-year survival drops from >90% in early-stage BC to ~32% for therapy-resistant subtypes such as triple-negative (TNBC), hormone-receptor–variable or resistant ER+, and Luminal B tumors. For these high-risk subtypes, molecular heterogeneity undermines targeted therapies, and clinical management still relies on maximum tolerable doses of systemic chemotherapy, causing severe dose-limiting toxicities. Moreover, the dense collagen-rich extracellular matrix (ECM) of solid tumors restricts intratumoral drug transport, motivating strategies that function across BC subtypes, overcome ECM barriers, and inform lowered clinical dosing. Bacteria-based cancer therapy (BBCT) with cancer-selective bacteria combines motility, self-replication, and on-board biosynthesis with the programmability of synthetic biology, enabling local release of therapeutic factors within the tumor microenvironment. Attenuated Salmonella Typhimurium VNP20009 (ST) exhibits ~10³-10⁴-fold tumor selectivity with respect to liver and spleen. It has a favorable clinical safety profile but remains inefficacious due to poor colonization. Our lab previously showed that ECM-targeting ST with collagenase secretion improved tumor penetration without gross collagen disruption, but at the cost of reduced bacterial fitness and motility. In this dissertation, we hypothesized that a fitness-restored, ECM-targeting ST enhances bacterial intratumoral transport and colonization, as well as chemotherapy penetration. We evaluated the engineered strains in perfused 3D tumor models that represent in vivo intratumor transport properties and investigated cancer cells-neutrophil interactions in presence of bacterial factors. First, we developed a high-motility, fitness-improved collagenase-expressing strain (HM-CEST ΔydcP) that preserves 100% motility under sub-cytotoxic chemotherapy. This strain improves intratumoral transport, and reduces spheroid viability and tumor migration relative to chemotherapy alone while maintaining tumor specificity and safety in preclinical murine models in vivo. Second, we validated a perfusion-enabled microfluidic spheroid platform that supports at least 14-day culture of murine TNBC and ER+ spheroids, enabling long-term BBCT screening. We demonstrated that perfused spheroids require lower chemotherapy doses than static cultures. Third, we biophysically characterized the crosstalk between BBCT, neutrophils, and Luminal B BC cells, demonstrating that neutrophils in the presence of bacteria supernatant suppress cancer cell growth, viability, and migration. Collectively, this work delivers a fitness-restored ECM-targeting Salmonella chassis, validates a perfusion-enabled 3D tumor-spheroid microphysiological platform, and develops a quantitative framework for neutrophil–bacteria–cancer cell interactions, contributing to the design pipeline for next-generation BBCT and supporting the long-term goal of safer, more affordable, lower-dose, and more broadly applicable therapies for therapy-resistant breast cancers."]},{"key":"dc:description.abstractgeneral","label":"General Abstract","values":["Breast cancer remains one of the most common and deadly cancers worldwide, with about 650,000 deaths reported in 2024. \"Breast cancer\" is not a single disease—some subtypes are much harder to treat than others. The hardest to treat forms often stop responding to hormone or targeted therapies and must be treated with toxic, high-dose chemotherapy that damages healthy cells in hair follicles, bone marrow, and the gut. This leads to hair loss, weakened immunity, gastrointestinal injury, and a high risk of infection, as well as long-term problems such as chronic fatigue, nerve damage, early menopause, and infertility, and increased risk of heart disease and secondary cancers. Despite aggressive chemotherapy, cancers are not always completely eliminated due to biological barriers present within tumors. Most solid cancers contain a dense, collagen-rich matrix that limits the penetration of drugs. Additionally, an immune cell population called neutrophils are \"recruited\" to the tumor to help it grow and shield cancer cells from chemotherapy. These barriers motivate new strategies that can work across breast cancer subtypes, overcome both physical and immune protection, and ultimately allow lower effective chemotherapy doses. Bacteria possess numerous anti-cancer properties. The overall goal of this doctoral dissertation builds on a clinically safe and cancer-targeting strain of Salmonella Typhimurium bacteria to self-propel in collagen-rich cancer environments while secreting a collagen-targeting enzyme. We show that in three-dimensional breast cancer models that mimic the dense tumor microenvironment, these bacteria penetrate more deeply and, when used briefly before chemotherapy, help a standard chemotherapeutic reduce tumor-cell survival and migration more effectively than chemotherapy alone, pointing toward the possibility of using lower, less toxic doses. To better reflect how tumors behave in the body, this dissertation validates a microfluidic \"tumor-on-a-chip\" device in which miniature breast tumors are contained and continuously supplied with nutrients and drugs. Through experiments on this platform, we show that improved treatment effects can be achieved with lower chemotherapy doses than in traditional no-flow cultures. Finally, this work examined how engineered bacteria influence neutrophils in a fast-growing breast cancer model, showing that cancer cells exposed to neutrophils plus bacterial effectors grow and move more slowly than untreated cancer cells. Together, this work shows how tumor-targeting bacteria and the body's own immune cells can be harnessed against therapy-resistant breast cancers, providing early evidence that such approaches could reduce our dependence on high-dose chemotherapy."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["ETD"]},{"key":"dc:title","label":"Title","values":["Engineering Tumor-Targeting Bacteria and Characterizing Their Interactions with Tumor Cells in Therapy-Resistant Breast Cancers"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Behkam, Bahareh"],"dc:contributor.committeemember":["Li, Liwu","Bortner, Michael J.","Johnson, Blake","Senger, Ryan S."],"dc:contributor.department":["Graduate School"],"dc:creator":["Saha, Binita"],"dc:date.accessioned":["2026-01-09T09:00:20Z"],"dc:date.available":["2026-01-09T09:00:20Z"],"dc:date.issued":["2026-01-08"],"dc:description.abstract":["Breast cancer (BC) accounts for one-third of malignancies among women in 157 countries and ~15% mortality among diagnosed cases, a burden projected to reach 1.1 million deaths per year by 2050. Five-year survival drops from >90% in early-stage BC to ~32% for therapy-resistant subtypes such as triple-negative (TNBC), hormone-receptor–variable or resistant ER+, and Luminal B tumors. For these high-risk subtypes, molecular heterogeneity undermines targeted therapies, and clinical management still relies on maximum tolerable doses of systemic chemotherapy, causing severe dose-limiting toxicities. Moreover, the dense collagen-rich extracellular matrix (ECM) of solid tumors restricts intratumoral drug transport, motivating strategies that function across BC subtypes, overcome ECM barriers, and inform lowered clinical dosing. Bacteria-based cancer therapy (BBCT) with cancer-selective bacteria combines motility, self-replication, and on-board biosynthesis with the programmability of synthetic biology, enabling local release of therapeutic factors within the tumor microenvironment. Attenuated Salmonella Typhimurium VNP20009 (ST) exhibits ~10³-10⁴-fold tumor selectivity with respect to liver and spleen. It has a favorable clinical safety profile but remains inefficacious due to poor colonization. Our lab previously showed that ECM-targeting ST with collagenase secretion improved tumor penetration without gross collagen disruption, but at the cost of reduced bacterial fitness and motility. In this dissertation, we hypothesized that a fitness-restored, ECM-targeting ST enhances bacterial intratumoral transport and colonization, as well as chemotherapy penetration. We evaluated the engineered strains in perfused 3D tumor models that represent in vivo intratumor transport properties and investigated cancer cells-neutrophil interactions in presence of bacterial factors. First, we developed a high-motility, fitness-improved collagenase-expressing strain (HM-CEST ΔydcP) that preserves 100% motility under sub-cytotoxic chemotherapy. This strain improves intratumoral transport, and reduces spheroid viability and tumor migration relative to chemotherapy alone while maintaining tumor specificity and safety in preclinical murine models in vivo. Second, we validated a perfusion-enabled microfluidic spheroid platform that supports at least 14-day culture of murine TNBC and ER+ spheroids, enabling long-term BBCT screening. We demonstrated that perfused spheroids require lower chemotherapy doses than static cultures. Third, we biophysically characterized the crosstalk between BBCT, neutrophils, and Luminal B BC cells, demonstrating that neutrophils in the presence of bacteria supernatant suppress cancer cell growth, viability, and migration. Collectively, this work delivers a fitness-restored ECM-targeting Salmonella chassis, validates a perfusion-enabled 3D tumor-spheroid microphysiological platform, and develops a quantitative framework for neutrophil–bacteria–cancer cell interactions, contributing to the design pipeline for next-generation BBCT and supporting the long-term goal of safer, more affordable, lower-dose, and more broadly applicable therapies for therapy-resistant breast cancers."],"dc:description.abstractgeneral":["Breast cancer remains one of the most common and deadly cancers worldwide, with about 650,000 deaths reported in 2024. \"Breast cancer\" is not a single disease—some subtypes are much harder to treat than others. The hardest to treat forms often stop responding to hormone or targeted therapies and must be treated with toxic, high-dose chemotherapy that damages healthy cells in hair follicles, bone marrow, and the gut. This leads to hair loss, weakened immunity, gastrointestinal injury, and a high risk of infection, as well as long-term problems such as chronic fatigue, nerve damage, early menopause, and infertility, and increased risk of heart disease and secondary cancers. Despite aggressive chemotherapy, cancers are not always completely eliminated due to biological barriers present within tumors. Most solid cancers contain a dense, collagen-rich matrix that limits the penetration of drugs. Additionally, an immune cell population called neutrophils are \"recruited\" to the tumor to help it grow and shield cancer cells from chemotherapy. These barriers motivate new strategies that can work across breast cancer subtypes, overcome both physical and immune protection, and ultimately allow lower effective chemotherapy doses. Bacteria possess numerous anti-cancer properties. The overall goal of this doctoral dissertation builds on a clinically safe and cancer-targeting strain of Salmonella Typhimurium bacteria to self-propel in collagen-rich cancer environments while secreting a collagen-targeting enzyme. We show that in three-dimensional breast cancer models that mimic the dense tumor microenvironment, these bacteria penetrate more deeply and, when used briefly before chemotherapy, help a standard chemotherapeutic reduce tumor-cell survival and migration more effectively than chemotherapy alone, pointing toward the possibility of using lower, less toxic doses. To better reflect how tumors behave in the body, this dissertation validates a microfluidic \"tumor-on-a-chip\" device in which miniature breast tumors are contained and continuously supplied with nutrients and drugs. Through experiments on this platform, we show that improved treatment effects can be achieved with lower chemotherapy doses than in traditional no-flow cultures. Finally, this work examined how engineered bacteria influence neutrophils in a fast-growing breast cancer model, showing that cancer cells exposed to neutrophils plus bacterial effectors grow and move more slowly than untreated cancer cells. Together, this work shows how tumor-targeting bacteria and the body's own immune cells can be harnessed against therapy-resistant breast cancers, providing early evidence that such approaches could reduce our dependence on high-dose chemotherapy."],"dc:description.degree":["Doctor of Philosophy"],"dc:format.medium":["ETD"],"dc:identifier.other":["vt_gsexam:45381"],"dc:identifier.uri":["https://hdl.handle.net/10919/140686"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["Creative Commons Attribution 4.0 International"],"dc:rights.uri":["http://creativecommons.org/licenses/by/4.0/"],"dc:subject":["Breast cancer","microphysiological 3D cancer models","combinatorial therapy","bacteria-based cancer therapy","extra cellular matrix targeting","immunotherapy"],"dc:title":["Engineering Tumor-Targeting Bacteria and Characterizing Their Interactions with Tumor Cells in Therapy-Resistant Breast Cancers"],"dc:type":["Dissertation"],"thesis:degree_discipline":["Macromolecular Science and Engineering"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:18:55Z"}