Investigating the Biophysical and Biochemical Influences of Stromal Cells on Anti-Cancer Drug Resistance within Bioengineered Tumor Microenvironment Models

癌症目前是美国第二大死因，每年夺走50多万美国人的生命。尽管癌症治疗研究取得了很大进展，但转移性癌症仍是致命的，这主要是由于它们对抗癌药物的耐药性。转移性癌症是指由另一个部位的原始生长的癌细胞传播而成的癌症。许多使用金标准动物模型和传统细胞培养方法开发的治疗策略往往因为没有考虑周围肿瘤微环境中癌细胞的复杂细胞和分子相互作用而失败。尽管利用生物工程肿瘤模型来研究癌症的不同方面取得了重大进展，但以往的研究都没有准确地重建复杂的肿瘤微环境，这些微环境由各种类型的间质细胞(结缔组织中的细胞)和细胞外基质(ECM)蛋白组成，这些蛋白对于确定每个微环境成分在抗癌耐药性中的作用至关重要。该项目的目标是利用能够容纳关键基质细胞和细胞外基质成分的生物工程肿瘤微环境模型，通过机械地研究基质细胞在抗癌耐药性中的作用，直接解决这一知识鸿沟。本研究的具体假设是，起源于肿瘤-间质串扰的物理和化学信号通路通过抑制药物穿透和激活癌细胞的代偿性耐药途径的组合机制而参与耐药。该研究计划在组织工程、3D成像和癌细胞生物学等多个科学学科之间带来了独特的协同效应。所获得的见解将导致对肿瘤间质串扰在抗癌耐药性中的作用的更全面的理解。肿瘤模型的独特功能还将克服现有方法的技术限制，从而使人们能够更好地了解抗癌治疗的有效性。关于教育影响，这项研究的多学科性质将在STEM领域吸引和培训新一代高中、本科生和研究生，并提供一个独特的机会，在生物工程师和癌症生物学家之间带来协同与合作。建议的计划还通过在线模块和出版物向科学界传播产生的基础知识。该项目侧重于使用与关键基质细胞、癌症相关成纤维细胞(CAF)和肿瘤相关巨噬细胞(TAM)结合的乳腺癌3D生物工程器官型肿瘤微环境模型，从机制上研究肿瘤微环境和基质细胞对抗癌药物耐药性的作用。该项目的主要假设是，源自肿瘤-间质串扰的理化信号信号导致不同的机制，这些机制最初促进间质重塑并抑制药物渗透，进而导致代偿性耐药途径的出现，这些途径共同导致化疗耐药。研究计划按两个目标组织。第一个目标是分析CAFS介导的间质间质结缔组织增生(纤维或结缔组织生长)在化疗耐药中的作用。将建立一个高密度的肿瘤间质模型，并与肿瘤生长过程中细胞外基质僵硬和重塑相关。一组抗纤维化药物将用于抑制间质纤维化，并将根据每种药物的反应评估基质重塑和僵硬。三维微流控平台将被用来测试化疗药物单独和联合抗纤维化药物的渗透性，药物分布曲线将与基质建模和硬度相关联。这些实验旨在验证这样一种假设，即化疗药物失败的主要机制是由于生物物理屏障和药物在基质中的渗透不足，无法到达肿瘤核心。第二个目标是从机制上研究来自基质CAF和TAMs的细胞信号在耐药中的协同影响。生物工程3D微流控平台将用于构建肿瘤间质模型，包括癌细胞、CAF和TAMs三种培养方式。已经用于治疗三阴性乳腺癌的一组化疗药物将被筛选出来，以评估单细胞水平的癌细胞侵袭以及对药物治疗的肿瘤存活和生长的反应。然后，对于在抑制肿瘤生长和侵袭方面表现出最低有效性的药物靶点，将利用RNA测序来评估癌症基因组学的变化，并确定导致耐药性的补偿途径。这些实验旨在检验这样一种假设，即由于细胞-细胞信号串扰，嵌入CAFS和TAMS共培养的基质中的综合反应比所有单个耐药线索的总和更明显。该项目的综合研究和生物学数据将有助于建立一个机制框架，说明在肿瘤-间质串扰的背景下，理化信号信号对抗癌药物耐药性的综合作用。总体而言，这些研究成果将推动与癌症生物学和抗癌耐药性相关的基础科学，并将为更广泛的生物制药芯片上肿瘤社区做出贡献。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Cancer is currently the second leading cause of death in the United States, claiming the lives of over half a million Americans annually. Although there has been a major thrust in cancer therapeutic research, metastatic cancers, i.e., cancers that have been formed by cancer cells that have been transmitted from an original growth at another site, are still deadly, largely due to their resistance to anti-cancer drugs. Many therapeutic strategies, developed using gold standard animal models and conventional cell culture methods, often fail due to not considering the complex cellular and molecular interactions of cancer cells within the surrounding tumor microenvironment. Despite significant advancements in the use of bioengineered tumor models to study different facets of cancer, none of the previous studies have accurately reconstructed the complex tumor microenvironment, composed of various types of stromal cells (cells in connective tissue) and the extracellular matrix (ECM) proteins that are crucial for determining the role of each microenvironmental component on anti-cancer drug resistance. The objective of this project is to directly addresses this knowledge gap by taking an innovative step forward to mechanistically study the role of stromal cells on anti-cancer drug resistance, utilizing bioengineered tumor microenvironment models that can accommodate key stromal cells and ECM components. The specific hypothesis of this study is that physical and chemical signaling cues, originating from tumor-stroma crosstalk, contribute to the drug resistance by a combinatorial mechanism of inhibiting drug penetration and activating compensatory drug resistance pathways in cancer cells. The research program brings a unique synergy between multiple scientific disciplines of tissue engineering, 3D imaging, and cancer cell biology. Insights gained will lead to more comprehensive understanding on the role of tumor-stroma crosstalk in anti-cancer drug resistance. The unique features of the tumor models will also overcome technical limitations of preexisting methods and thus allow better understanding of the effectiveness of anti-cancer therapeutics. With respect to the educational impact, the multidisciplinary nature of this study will engage and train a new generation of high school, undergraduate and graduate students in STEM fields and provide a unique opportunity to bring synergy and collaborations among bioengineers and cancer biologists. The proposed initiatives also promote dissemination of the generated fundamental knowledge to the scientific community through online modules and publications.This project focuses on mechanistically studying the role of tumor microenvironment and stromal cells on anti-cancer drug resistance using 3D bioengineered organotypic tumor microenvironmental models of breast cancer that are incorporated with key stromal cells, cancer associated fibroblasts (CAFs)and tumor associated macrophages (TAMs). The overarching hypothesis of the project is that the physiochemical signaling cues originating from tumor-stroma crosstalk lead to distinct mechanisms, which initially promote stromal remodeling and inhibit drug penetration and further results in emergence of compensatory drug-resistance pathways, which together lead to chemotherapeutic drug resistance. The research plan is organized under two objectives. The FIRST OBJECTIVE is to dissect the role of CAFs mediated stromal desmoplasia (growth of fibrous or connective tissue) on chemotherapeutic drug resistance. A high-density tumor-stroma model will be developed and characterized relative to ECM stiffness and remodeling during tumor growth. A panel of anti-fibrotic drugs will be used to inhibit stromal desmoplasia, and matrix remodeling and stiffness will be assessed in response to each drug. A 3D microfluidic platform will be used to test the penetration of chemotherapeutic drugs alone and in combination with the antfibrotic drugs, and the drug distribution profile will be correlated with stromal modeling and stiffness. The experiments are designed to test the hypothesis that the primary mechanism of failure of chemotherapeutic drugs is due to biophysical barriers and insufficient drug penetration within the stroma to reach the tumor core. The SECOND OBJECTIVE is to mechanistically study the synergistic influence of cellular signaling from stromal CAFs AND TAMs on drug resistance. The bioengineered 3D microfluidic platform will be used to construct a tumor-stroma model with a tri-culture of cancer cells, CAFs and TAMs. A panel of chemotherapeutic drugs that have been used in treating triple negative breast cancer will be screened to assess cancer cell invasion at the single cell level as well as tumor viability and growth in response to drug treatment. Then, in drug targets that have exhibited the lowest efficacy in inhibiting tumor growth and invasion, RNA sequencing will be utilized to assess alterations in cancer genomics and to identity compensatory pathways that lead to drug resistance. The experiments are designed to test the hypothesis that the integrated response within the stroma embedded with co-culture of CAFS and TAMs, due to cell-cell signaling crosstalk, is more pronounced than the sum of all the individual cues on drug resistance. The integrated studies and biological data from the project will enable the construction of a mechanistic framework on the comprehensive role of physiochemical signaling cues, in the context of tumor-stroma crosstalk, on anti-cancer drug resistance. Collectively, the research outcomes will advance fundamental science pertinent to cancer biology and anti-cancer drug resistance and will contribute to the broader biopharmaceutical tumor-on-chip communities.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.