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
Abstract 175: Production of cancer tissue-engineered microspheres for high-throughput screening
Abstract There is a need for new in vitro systems that enable pharmaceutical companies to collect more physiologically-relevant information on drug response in a low-cost and high-throughput manner. For this purpose, three-dimensional (3D) spheroidal models have been established as more effective than two-dimensional models. Current commercial techniques, however, rely heavily on self-aggregation of dissociated cells and are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of control over extracellular matrix components and heterogeneity in shape, size, and aggregate forming tendencies. In this study, we overcome these challenges by coupling tissue engineering toolsets with microfluidics technologies to create engineered cancer microspheres. Specifically, we employ biosynthetic hydrogels composed of conjugated poly(ethylene glycol) (PEG) and fibrinogen protein (PEG-Fb) to create engineered breast and colorectal cancer tissue microspheres for 3D culture, tumorigenic characterization, and examination of potential for high-throughput screening (HTS). MCF7 and MDA-MB-231 cell lines were used to create breast cancer microspheres and the HT29 cell line and cells from a stage II patient-derived xenograft (PDX) were encapsulated to produce colorectal cancer (CRC) microspheres. Using our previously developed microfluidic system, highly uniform cancer microspheres (intra-batch coefficient of variation (CV) ≤ 5%, inter-batch CV < 2%) with high cell densities (>20×106 cells/ml) were produced rapidly, which is critical for use in drug testing. Encapsulated cells maintained high viability and displayed cell type-specific differences in morphology, proliferation, metabolic activity, ultrastructure, and overall microsphere size distribution and bulk stiffness. For PDX CRC microspheres, the percentage of human (70%) and CRC (30%) cells was maintained over time and similar to the original PDX tumor, and the mechanical stiffness also exhibited a similar order of magnitude (103 Pa) to the original tumor. The cancer microsphere system was shown to be compatible with an automated liquid handling system for administration of drug compounds; MDA-MB-231 microspheres were distributed in 384 well plates and treated with staurosporine (1 μM) and doxorubicin (10 μM). Expected responses were quantified using CellTiter-Glo® 3D, demonstrating initial applicability to HTS drug discovery. PDX CRC microspheres were treated with Fluorouracil (5FU) (10 to 500 μM) and displayed a decreasing trend in metabolic activity with increasing drug concentration. Providing a more physiologically relevant tumor microenvironment in a high-throughput and low-cost manner, the PF hydrogel-based cancer microspheres could potentially improve the translational success of drug candidates by providing more accurate in vitro prediction of in vivo drug efficacy. Citation Format: Elizabeth A. Lipke, Wen J. Seeto, Yuan Tian, Mohammadjafar Hashemi, Iman Hassani, Benjamin Anbiah, Nicole L. Habbit, Michael W. Greene, Dmitriy Minond, Shantanu Pradhan. Production of cancer tissue-engineered microspheres for high-throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 175.
more »
« less
- Award ID(s):
- 2141205
- NSF-PAR ID:
- 10400546
- Date Published:
- Journal Name:
- Cancer Research
- Volume:
- 82
- Issue:
- 12_Supplement
- ISSN:
- 1538-7445
- Page Range / eLocation ID:
- 175 to 175
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Coordination of clinically employed bisphosphonate, risedronate (RISE), to bioactive metals, Ca2+, Mg2+, and Zn2+, allowed the formation of bisphosphonate-based coordination complexes (BPCCs). Three RISE-based BPCCs, RISE-Ca, RISEMg, and RISE-Zn, were produced, and their structures were elucidated by single crystal X-ray difraction. Interestingly, the addition of an auxiliary ligand, etidronic acid (HEDP), resulted in the recrystallized protonated form of the ligand, H-RISE. The pH-dependent structural stability of the RISE-based BPCCs was measured by means of dissolution profles under neutral and acidic simulated physiological conditions (PBS and FaSSGF, respectively). In comparison to RISE (Actonel), the complexes showed a lower equilibrium solubility (∼70−85% in 18−24 h) in PBS, while a higher equilibrium solubility (∼100% in 3 h) in acidic media. The results point to the capacity to release this BP in a pH-dependent manner from the RISE-based BPCCs. Subsequently, the particle size of RISE-Ca was reduced, from 300 μm to ∼350 d.nm, employing the phase inversion temperature (PIT)-nanoemulsion method, resulting in nano-Ca@RISE. Aggregation measurements of nano-Ca@RISE in 1% fetal bovine serum (FBS):H2O was monitored after 24, 48, and 72 h to study the particle size longevity in physiological media, showing that the suspended material has the potential to maintain its particle size over time. Furthermore, binding assays were performed to determine the potential binding of nano-Ca@RISE to the bone, where results show higher binding (~1.7×) for the material to hydroxyapatite (HA, 30%) when compared to RISE (17%) in 1 d. The cytotoxicity efects of nano-Ca@RISE were compared to those of RISE against the human breast cancer MDA-MB-231 and normal osteoblast-like hFOB 1.19 cell lines by dose−response curves and relative cell viability assays in an in vitro setting. The results demonstrate that nano-Ca@RISE signifcantly decreases the viability of MDA-MB-231 with high specifcity, at concentrations ∼2−3× lower than the ones reported employing other third-generation BPs. This is supported by the fact that when normal osteoblast cells (hFOB 1.19), which are part of the tissue microenvironment at metastatic sites, were treated with nano-Ca@RISE no signifcant decrease in viability was observed. This study expands on the therapeutic potential of RISE beyond its antiresorptive activity through the design of BPCCs, specifcally nano-Ca@RISE, that bind to the bone and degrade in a pH-dependent manner under acidic conditionsmore » « 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
-
Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor-homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.more » « less
-
Senescence is a potent tumor-suppressive mechanism that irreversibly arrests the growth of damaged cells. However, senescent cells that accumulate in tissues eventually develop a senescence-associated secretory phenotype (SASP) that alters the microenvironment to promote cancer. Paracrine factors in the SASP may also contribute to the formation of rare giant polyploidal cancer cells (GPCCs). A single-cell mechanical approach was used to profile cytoskeletal and nuclear mechanics, morphology, motility, and adhesion for breast cancer cells treated with conditioned media from senescent fibroblasts. Our study showed that a small but significant population of MDA-MB-231 breast cancer cells (less than 5%) treated with conditioned media from senescent LF-1 fibroblasts develop an enlarged morphology, chromosomal instability, and polyploidy, a phenotype associated with GPCCs. Although GPCCs are highly invasive and chemoresistant, little is known about their biophysical properties. First, we developed a method for identifying the small subpopulation of GPCCs in a heterogeneous population of cancer cells based on increased nuclear area and confirmed that GPCCs are more resistant to paclitaxel than normal-size MDA-MB-231 cells (NCCs). We then compared critical biophysical properties of NCCs and GPCCs, including cytoskeletal and nuclear mechanics, cell and nuclear morphology, motility, and adhesion. Cells were stained for cytoskeletal proteins actin, tubulin, and vinculin. Cytoskeletal organization was dramatically altered in GPCCs compared to NCCs. GPCCs displayed more disorganized microtubule structure, dense actin stress fibers, and mature focal adhesions. Intracellular particle tracking microrheology was used to measure cytoskeletal and nuclear mechanics. These studies demonstrated that although GPCCs are thought to be highly invasive cancer cells, they are inherently stiffer than NCCs, in terms of both their cytoskeletal and nuclear mechanics. This was surprising since more invasive cancer cells are often more compliant than less invasive cancer cells. This result may be in part to the ability for GPCCs to behave like activated stromal cells that stiffen in the tumor; we confirmed that GPCCs display similar adhesive behavior as activated stromal cells. To determine how mechanics correlates with cell migration, we used time-lapse nuclear tracking to measure cell motility. The average cell speed was higher for NCCs than for GPCCs; however, GPCCs moved longer distances over time because their motion was more directional. These findings highlight the unusual biophysical behavior of GPCCs. To develop pharmacologic tools that target GPCCs, it is imperative to understand their biophysical properties.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1158/1538-7445.AM2022-175
