The development of physiologically relevant in vitro colorectal cancer (CRC) models is vital for advancing understanding of tumor biology. Although CRC patient-derived xenografts (PDX) recapitulate key patient tumor characteristics and demonstrate high concordance with clinical outcomes, the use of this in vivo model is costly and low-throughput. Here we report the establishment and in-depth characterization of an in vitro tissue-engineered CRC model using PDX cells. To form the 3D engineered CRC-PDX (3D-eCRC-PDX) tissues, CRC PDX tumors were expanded in vivo, dissociated, and the isolated cells encapsulated within PEG-fibrinogen hydrogels. Following PEG-fibrinogen encapsulation, cells remain viable and proliferate within 3D-eCRC-PDX tissues. Tumor cell subpopulations, including human cancer and mouse stromal cells, are maintained in long-term culture (29 days); cellular subpopulations increase ratiometrically over time. 3D-eCRC-PDX tissues mimic the mechanical stiffness of originating tumors. ECM protein production by cells in the 3D-eCRC-PDX tissues resulted in approximately 57% of proteins observed in the CRC-PDX tumors also being present in the 3D-eCRC-PDX tissues on Day 22. Furthermore, we show congruence in enriched gene ontology molecular functions and Hallmark gene sets in 3D-eCRC-PDX tissues and CRC-PDX tumors compared to normal colon tissue, while prognostic Kaplan-Meier plots for overall and relapse free survival did not reveal significant differences between CRC-PDX tumors and 3D-eCRC-PDX tissues. Our results demonstrate high batch-to-batch consistency and strong correlation between our in vitro tissue-engineered PDX-CRC model and the originating in vivo PDX tumors, providing a foundation for future studies of disease progression and tumorigenic mechanisms.

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Prostate cancer (PC) currently represents 7.5% of all new cancer cases; notably, the 5-year relative survival rate drops from 100% in localized cases to 30.2% in patients who present with metastases. There are no curative therapies for metastatic PC, and most men develop serial resistance to androgen suppression, resulting in a more aggressive disease state that is much more difficult to mitigate. Fibroblasts have been implicated in cancer progression and are thought to intravasate alongside circulating tumor cells and prime metastatic sites for tumor growth. Our understanding of the precise mechanisms by which they contribute to PC, however, is relatively underdeveloped in comparison to other solid cancer types. Here, we report a three-dimensional (3D) engineered prostate cancer tissue (EPCaT) model comprised of PC-3 castration-resistant (CRPC) or LNCaP androgen-dependent (ADPC) PC cell lines in direct coculture with BJ-5ta fibroblasts. By specifically isolating this cell-cell interaction within a bioinspired poly(ethylene glycol)-fibrinogen (PF) matrix, our EPCaT model introduces the ability to monitor coculture-driven changes at a tissue, cellular, and transcriptomic level. Temporal variations in EPCaT growth, cell and colony morphology, cell populations, and cell-mediated remodeling of the PF matrix were assessed. Changes in bulk transcriptomic expression were also quantified and differentially expressed genes (DEGs) were evaluated between CRPC and ADPC mono- and coculture conditions. Finally, to evaluate the clinical significance of our findings, EPCaTs were evaluated against normal and primary tumor tissue transcriptomic data acquired from the Cancer Genome Atlas (TCGA). In comparison to monoculture EPCaTs, both CRPC- and ADPC-fibroblast coculture conditions resulted in an increase in the number of proliferative cells, morphological features of cancer cell migration, and cell-mediated remodeling of the PF matrix, all of which suggest a more aggressive cell phenotype. DEG and gene ontology analysis revealed coculture-driven changes in genes associated with important tumorigenic processes including ECM organization, angiogenesis, and epithelial cell proliferation and migration. Interestingly, fibroblast coculture had a significantly larger impact on the ADPC transcriptome in comparison to CRPC, suggesting that fibroblasts could play an elevated role in less aggressive disease states. Notable DEGs in ADPC coculture that were also clinically significant in the TCGA tumor versus normal comparison included an overexpression of OR51E2 which has been shown to increase epithelial cell proliferation and participate in the ADPC to CRPC switch, thus exacerbating PC progression. Future studies will augment the pathophysiological relevance of our EPCaT model by including patient-isolated cancer-associated fibroblasts from recurring and non-recurring patients. Citation Format: Nicole L. Habbit, Benjamin Anbiah, Joshita Suresh, Yuan Tian, Luke S. Anderson, Megan L. Davies, Iman Hassani, Taraswi Mitra Ghosh, Balabhaskar Prabhakarpandian, Robert D. Arnold, Elizabeth A. Lipke. Elucidating the role of fibroblasts in CRPC and ADPC progression using 3D engineered prostate cancer tissues [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 3856.

To investigate the ratiometric role of fibroblasts in prostate cancer (PCa) progression, this work establishes a matrix‐inclusive, three‐dimensional engineered prostate cancer tissue (EPCaT) model that enables direct coculture of neuroendocrine‐variant castration‐resistant (CPRC‐ne) or androgen‐dependent (ADPC) PCa cells with tumor‐supporting stromal cell types. Results show that the inclusion of fibroblasts within CRPC‐ne and ADPC EPCaTs drives PCa aggression through significant matrix remodeling and increased proliferative cell populations. Interestingly, this is observed to a much greater degree in EPCaTs formed with a small number of fibroblasts relative to the number of PCa cells. Fibroblast coculture also results in ADPC behavior more similar to the aggressive CRPC‐ne condition, suggesting fibroblasts play a role in elevating PCa disease state and may contribute to the ADPC to CRPC‐ne switch. Bulk transcriptomic analyses additionally elucidate fibroblast‐driven enrichment of hallmark gene sets associated with tumorigenic progression. Finally, the EPCaT model clinical relevancy is probed through a comparison to the Cancer Genome Atlas (TCGA) PCa patient cohort; notably, similar gene set enrichment is observed between EPCaT models and the patient primary tumor transcriptome. Taken together, study results demonstrate the potential of the EPCaT model to serve as a PCa‐mimetic tool in future therapeutic development efforts.This article is protected by copyright. All rights reserved

Biomimetic tissue engineered microfluidic cancer models offer a higher degree of spatial, temporal and structural precision in controlling the physical parameters and component characteristics of the native tumor microenvironment (TME). Current models working to establish a biomimetic in vitro breast TME are limited by their ability to recapitulate various degrees of in vivo complexities and poor correlation of the diffusional gradients of oxygen, nutrients and anti-cancer drugs. To establish a model and address these challenges, we have used poly(ethylene glycol)-fibrinogen (PEG-Fb) as our biomimetic material to engineer 3D breast tumor tissues and recapitulate the mechanical stiffness of core, midpoint and peripherial zones of the native tumor in a vascularized microfluidic chip. To assess the mechanical stiffness, the in vivo breast tumor (MDA-MB-231 flank xenograft in Athymic nude mice) and engineered tumor constructs were subjected to parallel plate compression test using Cell Scale Microsquisher and the resulting force versus displacement data was acquired to calculate Young’s modulus. Tumor mimetic (“high perfusion chip” (HPC) and “low perfusion chip” (LPC), differ with respect to the vascular network surrounding their respective primary and secondary tumor compartments were used in this study. Breast cancer-associated endothelial cells (hBTEC) were seeded in the vascular network and allowed to form a lumen. Metastatic breast cancer cells MDA-MB-231/ human foreskin fibroblast BJ5ta (ATCC) cells were mixed with polymer precursor solution containing PEG-Fb and Eosin Y. The precursor was loaded into the primary tumor compartment and cross-linked for 2 minutes under visible light. Stiffness was modulated by adding poly(ethylene glycol) diacrylate (PEGDA) to the polymer precursor for recapitulating the different zones of the in vivo tumor. hBTEC media was perfused through the endothelial cell networks were continuously monitored for cell behavior and metastasis. In vivo breast tumor stiffness at core, midpoint and periphery was found to be within the range of the 3D engineered breast tumor tissues with time in culture through day 29. In the vascularized microfluidic chip, cell laden biomaterial was incorporated, the cancer cells were observed to undergo key events of the TME such as intravasation, circulating tumor cells in the endothelial vascular channel, adherence and migration to the secondary chamber resulting in metastasis. In the native TME there are regional differences in drug diffusion; TRITC dextran (4.4 kDa) was administered at a constant flow rate through the chips’ vascularized networks and found to have vascular network geometry and engineered tumor construct stiffness dependent differences in diffusion into the primary tumor chamber, mimicking the in vivo phenomena. Citation Format: Benjamin Anbiah, Iman Hassani, Nicole Habbit, Lani Jasper, Deborah Ramsay, Balabhaskar Prabhakarpandian, Robert Arnold, Elizabeth Lipke. In vivo breast tumor stiffness and vascular drug delivery recapitulated in a microfluidic tumor-on-a-chip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 999.

Colorectal cancer (CRC) is the third-most leading cause of cancer-related deaths in the United States. To advance the understanding of CRC tumor progression, models which mimic the tumor microenvironment (TME) and have translatable study outcomes are urgently needed. CRC patient-derived xenografts (PDXs) are promising tools for their ability to recapitulate tumor heterogeneity and key patient tumor characteristics, such as molecular characteristics. However, as in vivo models, CRC PDXs are costly and low-throughput, which leads to a need for equivalent in vitro models. To address this need, we previously established an in vitro model using a tissue engineering toolset with CRC PDX cells. However, it is unclear whether tissue engineering has the capacity to maintain patient- and/or cancer stage-specific tumor heterogeneity. To address this gap, we employed three PDX tumor lines, originated from stage II, III-B, and IV CRC tumors, in the formation of 3D engineered CRC PDX (3D-eCRC-PDX) tissues and performed an in-depth comparison between the 3D-eCRC-PDX tissues and the original CRC-PDX tumors. To form the tissues, CRC-PDX tumors were expanded in vivo and dissociated. The isolated cells were encapsulated within poly(ethylene glycol)-fibrinogen hydrogels and remained viable and proliferative post encapsulation over the course of 29 days in culture. To gain molecular insight into the maintenance of PDX line stage heterogeneity, we performed a transcriptomic analysis using RNA seq to determine the extent to which there were similarities and differences between the CRC-PDX tumors and the 3D-eCRC-PDX tissues. We observed the greatest correspondence in overlapping differentially expressed human genes, gene ontology, and Hallmark gene set enrichment between the 3D-eCRC-PDX tissues and CRC-PDX tumors in the stage II PDX line, while the least correspondence was observed in the stage IV PDX line. The Hallmark gene set enrichment from murine mapped RNA seq transcripts was PDX line-specific which suggested that the stromal component of the 3D-eCRC-PDX tissues was maintained in a PDX line-dependent manner. Consistent with our transcriptomic analysis, we observed that tumor cell subpopulations, including human proliferative (B2M+Ki67+) and CK20+ cells, remained constant for up to 15 days in culture even though the number of cells in the 3D-eCRC-PDX tissues from all three CRC stages increased over time. Yet, tumor cell subpopulation differences in the stage IV 3D-eCRC-PDX tissues were observed starting at 22 days in culture. Overall, our results demonstrate a strong correlation between our in vitro 3D-eCRC-PDX models and the originating in vivo CRC-PDX tumors, providing evidence that these engineered tissues may be capable of mimicking patient- and/or cancer stage-specific heterogeneity. Citation Format: Yuan Tian, Iman Hassani, Benjamin Anbiah, Bulbul Ahmed, William Van Der Pol, Elliot J. Lefkowitz, Peyton C. Kuhlers, Nicole L. Habbit, Martin J. Heslin, Elizabeth A. Lipke, Michael W. Greene. Transcriptomic analysis of a 3D engineered cancer model recapitulating stage-dependent heterogeneity in colorectal PDX tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4567.

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.

Strong epidemiological evidence links obesity to an increased risk of colorectal cancer (CRC). However, the precise molecular mechanisms underlying such an association have not been fully elucidated, partly due to the lack of physiologically relevant models. Here, we established a novel PEG-fibrinogen-based engineered tumor model using patient-derived xenograft (PDX) CRC co-cultured with 3T3-L1 adipocytes and an orthotopically implanted PDX CRC model of obesity. The PDX CRC cells were isolated from PDXs propagated subcutaneously in NOD-SCID mice and encapsulated within a biomimetic polymer, PEG-fibrinogen, to create 3D engineered PDX CRC tumors (3DePCCTs) or cultured in a standard 2D manner. We compared cell viability, colony area, and cell subpopulations within the 3DePCCTs and stiffness of the 3DePCCTs to the in vivo propagated tumors. A stage IV CRC tumor was employed in vivo and in vitro to study obesity-promoted tumor growth. Responsiveness of the 3DePCCTs to the growth promoting effects of obesity was investigated through continuous co-culture with insulin sensitive (IS) and resistant (IR) (treated with TNFα and 1% hypoxia) 3T3-L1 adipocytes (adipocytes replaced every 72 hrs). The responsiveness of the stage IV CRC PDX line was validated using an orthotopically implanted CRC model of obesity in which Rag1tm1Mom mice were fed a high fat Western diet + 4% sugar water (HFWD+S) to induce obesity or a chow diet to maintain a lean phenotype. The cells within the 3DePCCTs remained viable and the PDX-line dependent differential growth of tumor colony area within the 3DePPCTs recapitulated line-dependent difference in in vivo tumor growth. Based on flow cytometry, human (70±3%) and Cytokeratin 20+ (31±1) cell subpopulations within the 3DePCCTs were similar to the original in vivo tumor tissue and maintained over time, whereas supporting mouse stromal cells took over 2D cultured cells (n=3 batches). Stiffness of the 3DePCCTs was within the range of the in vivo tumor tissue stiffness (0.3 to3.6 KPa, n=3 tissues). In vitro adipocytes maintained IR for at least 72 hrs based on significantly higher MCP1 (at least 10 folds) and lower GLUT4 (maximally 0.1 fold) (n=3 batches). The in vitro coculture model revealed a significantly (p < 0.05) greater number of cancer cell colonies within 3DePCCTs after 8 days of co-culture with IR adipocytes compared to IS adipocytes and this difference increased through day 29. In the in vivo model, a significant (p < 0.05) greater than 2-fold increase in weight of PDX CRC tumors grown in mice fed the HFWD+S diet was observed. We have established the ability to maintain PDX CRC cells in 3D culture long-term (29 days) and generated a novel in vitro obesity-mimetic engineered tumor model that recapitulated the growth promoting effects of the in vivo orthotopic, IR tumor microenvironment and could be used to examine mechanistic questions and therapeutic targets. Citation Format: Iman Hassani, Benjamin Anbiah, Bulbul Ahmed, Nicole L. Habbit, Michael W. Greene, Elizabeth A. Lipke. In vitro recapitulation of in vivo obesity-promoted colorectal cancer growth using a patient-derived xenograft engineered tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2844.