Tumor microenvironment is a complex niche consisting of cancer cells and stromal cells in a network of extracellular matrix proteins and various soluble factors. Dynamic interactions among cellular and non-cellular components of the tumor microenvironment regulate tumor initiation and progression. Fibroblasts are the most abundant stromal cell type and dynamically interact with cancer cells both in primary tumors and in metastases. Cancer cells activate resident fibroblasts to produce and secrete soluble signaling molecules that support proliferation, migration, matrix invasion, and drug resistance of cancer cell and tumor angiogenesis. In recent years, various forms of three-dimensional tumor models have been developed to study tumor–stromal interactions and to identify anti-cancer drugs that block these interactions. There is currently a technological gap in development of tumor models that are physiologically relevant, scalable, and allow convenient, on-demand addition of desired components of the tumor microenvironment. In this review, we discuss three studies from our group that focus on developing bioengineered models to study tumor-stromal signaling. We will present these studies chronologically and based on their increasing complexity. We will discuss the validation of the models using a CXCL12-CXCR4 chemokine-receptor signaling present among activated fibroblasts and breast cancer cells in solid tumors, highlight the advantages and shortcomings of the models, and conclude with our perspectives on their applications. Impact statement Tumor stroma plays an important role in progression of cancers to a fatal metastatic disease. Modern treatment strategies are considering targeting tumor stroma to improve outcomes for cancer patients. A current challenge to develop stroma-targeting therapeutics is the lack of preclinical physiologic tumor models. Animal models widely used in cancer research lack human stroma and are not amenable to screening of chemical compounds for cancer drug discovery. In this review, we outline in vitro three-dimensional tumor models that we have developed to study the interactions among cancer cells and stromal cells. We describe development of the tumor models in a modular fashion, from a spheroid model to a sophisticated organotypic model, and discuss the importance of using correct physiologic models to recapitulate tumor-stromal signaling. These biomimetic tumor models will facilitate understanding of tumor-stromal signaling biology and provide a scalable approach for testing and discovery of cancer drugs.
more »
« less
Evaluating Nanoparticle Penetration In Tumor Spheroids
Introduction: The lack of an appropriate in vitro model of the tumor microenvironment is one of the largest
obstacles in evaluating preclinical cancer drug screenings.1 Cancer cell monolayers do not effectively mimic the limited drug penetration properties of the complex tumor structures found in cancer patients. 3-D multicellular tumor spheroids (MCTS) serve as a more effective model as they better resemble cancer in structure as well as limited drug penetration. In our experiments, we created heterospheroids composed of 4T1 breast tumor cells and 3T3 fibroblasts, as well as homospheroids of each cell type. Tumors feature stromal and extracellular matrix components in addition to cancer cells in ratios that vary between different types of cancer. Fibroblasts are the major component of cancer stroma as well as producers of extracellular matrix. Since heterospheroids feature 3T3 fibroblasts, they may better model the diverse tumor microenvironment.2 We also synthesized fluorescent PLGA nanoparticles that were added to our spheroid cultures. Using confocal microscopy and ImageJ’s fluorescence measuring tools, we qualitatively and quantitatively evaluated the drug penetration properties of our spheroids.
more »
« less
- Award ID(s):
- 1757885
- NSF-PAR ID:
- 10138551
- Date Published:
- Journal Name:
- 2019 BMES Conference Proceedings - REU Abstract Accepted Poster
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Cells in vivo generate mechanical traction on the surrounding 3D extracellular matrix (ECM) and neighboring cells. Such traction and biochemical cues may remodel the matrix, e.g., increase stiffness, which, in turn, influences cell functions and forces. This dynamic reciprocity mediates development and tumorigenesis. Currently, there is no method available to directly quantify single-cell forces and matrix remodeling in 3D. Here, we introduce a method to fulfill this long-standing need. We developed a high-resolution microfabricated sensor that hosts a 3D cell-ECM tissue formed by self-assembly. This sensor measures cell forces and tissue stiffness and can apply mechanical stimulation to the tissue. We measured single and multicellular force dynamics of fibroblasts (3T3), human colon (FET) and lung (A549) cancer cells, and cancer-associated fibroblasts (CAF05) with 1-nN resolution. Single cells show notable force fluctuations in 3D. FET/CAF coculture system, mimicking cancer tumor microenvironment, increased tissue stiffness by three times within 24 hours.more » « less
-
Age is a leading risk factor for developing breast cancer. This may be in part to the time required for acquiring sufficient cancer mutations; however, stromal cells that accumulate in tissues and undergo senescence eventually develop a senescence-associated secretory phenotype that alters the microenvironment to promote cancer. Our focus is on mesenchymal stem cells (MSCs) – stromal cells recruited to tumors due to their natural tropism for inflammatory tissues; MSCs have been shown to enhance the metastatic potential of tumor cells through direct interactions or paracrine signaling within the tumor. In the tumor, MSCs can differentiate into carcinoma-associated fibroblasts that play a central role in tumor growth and matrix remodeling. We recently investigated the molecular and mechanical differences in pre- and post- senescent MSCs and how their interactions with MDA-MB-231 breast cancer cells contribute to malignancy. Our data show post-senescent MSCs are larger and less motile, with more homogeneous mechanical properties than pre-senescent MSCs. In-depth omics analysis revealed differentially regulated genes and peptides including factors related to inflammatory cytokines, cell adhesion to the extracellular matrix, and cytoskeletal regulation. A 3D co-culture model was used to assess the effects of pre- and post- senescent MSCs on collagen matrix remodeling. Although post-senescent MSCs were far less motile than pre-senescent MSCs and less contractile with the matrix, they profoundly altered matrix protein deposition and crosslinking, which resulted in local matrix stiffening effects. Post-senescent MSCs also induced an invasive breast cancer cell phenotype, characterized by increased proliferation and invasion of breast cancer cells. This invasive breast cancer cell behavior was further amplified when MDA-MB-231 was co-cultured with a mixture of pre- and post- senescent MSCs; this result was attributed to matrix remodeling and soluble factor secretion effects of post-senescent MSCs, which enhanced the migration of pre-senescent MSCs allowing them to form tracks in the collagen network for cancer cells to follow. Finally, molecular inhibitors targeting actomyosin contractility and adhesion were used to alter MSC interactions with breast cancer cells. Actin depolymerizing agent and focal adhesion kinase inhibitor were most efficient and completely able to block the effects of post-senescent MSCs on MDA-MB-231 invasion in collagen gels. This comprehensive approach can be used to identify molecular pathways regulating heterotypic interactions of post-senescent MSCs with other cells in the tumor. Furthermore, the local matrix stiffening effect of post-senescent MSCs may play a critical role in breast cancer progression.more » « less
-
more » « less
Abstract 3D bioprinting improves orientation of
in vitro tumor models by offering layer by layer positioning of cancer cells and cancer associated fibroblasts (CAFs) which can replicate tumor microenvironment. Aim of this study was to develop a sodium alginate -gelatin (SA-GL) hydrogel by optimizing rheological parameters to print non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) cells and lung CAFs co-cultures. SA-GL hydrogels were prepared, and rheological properties were evaluated. Both the cells were mixed with the hydrogel and printed using INKREDIBLE bioprinter. Hydrogels prepared with 3.25% and 3.5% (w/v) SA and 4% (w/v) GL showed higher printability and cell viability. A significant decline in viscosity with shear rate was observed in these hydrogels suggesting the shear thinning property of hydrogels. Spheroid size distribution after 15 days was in the diameter range of 50–1100 µm. Up-regulation of vimentin, α-SMA and loss of E-cadherin in co-culture spheroids confirmed cellular crosstalk. This study demonstrates that rheological optimization of SA-GL hydrogel enhances printability and viability of NSCLC PDX and CAF co-culture which allows 3D co-culture spheroid formation within the printed scaffold. Therefore, this model can be used for studying high throughput drug screening and other pre-clinical applications. -
Abstract Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune–cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8 + T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune–cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune–cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Conference Paper:
- The DOI is not currently available.
