PROJECT SUMMARY/ABSTRACT
The tumor microenvironment is known for its complexity, both in its content as well as its dynamic nature. It
consists of various immune cells, stromal fibroblasts, extracellular matrix (ECM) components often associated
with wound healing and inflammation, blood and lymph vessels, and various other cell types like endothelial
progenitor cell. During cancer initiation and progression, these cells interact in a highly complex environment,
which is not trivial to understand using two-dimensional models of tumor cell culture. Several advances in tissue
engineering have allowed more physiologically-relevant in vitro tumor microenvironment models to be
developed. Three-dimensional (3D) tumor spheroid cultures serve as powerful tools for dissecting the roles of
various biochemical and biophysical cues in carcinoma initiation and progression. Although currently utilized
microfluidic-based approaches enable cell-cell interactions in 3D, they lack the ability to control the organization
of multiple cell types and cannot build complex 3D architectures, which typically requires manual manipulations
of cells and biomaterials. The bioprinting approach proposed in this study will establish a biomimetic model
platform that can be perfused through integration of built-in channels and recapitulates the cancer
microenvironment. This model will advance our understanding of the fundamental interactions taking place
between immune cells and cancer cells during cancer initiation and progression, and thereby reveal how these
interactions can alleviate the progression of cancer. We hypothesize that cellular and molecular interactions
between immune and tumor cells in 3D environments will differ from monolayer cultures and more closely
recapitulate the in vivo state. To test this hypothesis, Specific Aim 1 will use bioprinting approaches to develop
3D models of breast cancer and human engineered T cells that can be activated by tumor cells within this in vitro
microenvironment. In Specific Aim 2, we will determine the transcriptional impact of events within the 3D tumor-
immune cell microenvironment compared to monolayer cultures through single cell RNA-sequencing approach.
We will also assess the impact of different T cell subsets and myeloid lineage cells, such as macrophages and
dendritic cells, on structural organization, angiogenic potential, metastatic behavior and cytotoxicity within the
3D tumor microenvironment. Further, we will test several approaches to enhance T cell responses to tumors in
3D bioprinted tissues, by CRISPR/Cas9 editing of antibody neutralization of T cell inhibitory molecules such as
PD1 and CTLA-4. In this regard, we have formed a complementary collaboration that merges essential domain
knowledge in human immunology, cell engineering and 3D bioprinting, with the depth necessary to propel the
proposed work towards meaningful advances that would otherwise not be possible. Successful completion of
the proposed work is anticipated to give rise to an integrated mechanistic framework revealing the complex
interactions between immune cells and tumors in 3D biomimetic environment and thereby provide a novel
understanding of how immune cells impact cancer intravasation and metastasis.