Poster S17 - Micro-milled high-throughput force sensor array for in situ mechanical testing of tumors-on-a-chip

Introduction: Facile and precise measurement of cellular forces within three-dimensional (3D) extra-cellular matrices (ECM) has long been a challenge. Addressing this critical gap, we introduced nano-Newton resolution sensor to quantify i) force of a single (or very few) cell(s) in 3D matrices, and ii) quantify the stiffness of the cell-matrix environment as function of time in order to assess matrix remodeling by the cells [1]. The sensor bypasses the necessity of constitutive relations and uses force balance to quantify traction forces generated by very small cell clusters in analytically difficult matrices [1]. While this novel method has successfully been implemented in laboratories for research purposes, it being relatively expensive and complicated precludes widespread adoption of the technique for clinical applications. In order to augment the sensors adaptability and ease of operation, we fabricated a high-throughput sensor array made with PDMS using low-cost micro-milled acrylic molds.
This innovative approach offers a multitude of advantages over the earlier versions. Firstly, it allows for the simultaneous generation of numerous samples within a single dish, facilitating the exploration of diverse culture conditions with ease. Secondly, its straightforward fabrication process markedly reduces production costs compared to traditional microfabrication techniques, facilitating access to advanced biomechanical tools. Thirdly, the versatility of the system opens new avenues for applications beyond fundamental research, including its potential integration into hospitals, paving the way for biomechanics-based personalized medicine. With its transformative capabilities, our high-throughput sensor array promises to propel forward the frontiers of cellular biomechanics and facilitating advancements in therapeutic interventions.

Materials and

Methods: A simplified schematic shows the mechanism of the sensor (Fig. 1). Briefly, a capillary bridge is formed with cell-ECM (rat-tail collagen I) mixture between two grips connected to force sensing springs (Fig. 1A). If force by the cells stretch the spring by df, we can determine the force from F = Kf * df ; Kf being the spring constant (Fig. 1A). Stretching/compressing allows stiffness measurement of the tissue construct. Unit sensor, made of PDMS, consists of beams that function as springs (Fig. 1B).
To fabricate the mold of a high-throughput sensor array, an acrylic sheet was drilled to 200 um depth with a 75-um end-mill on a Bulkman 3D Ultimate Bee CNC machine (Fig. 2A). Mold for the top and bottom layer was cut with a 500-um end-mill. The sensor, top and bottom layers were prepared by pouring PDMS and curing at 60 °C. Finally, the array is prepared by sticking these layers to a glass slide (Fig. 2B,C).
With an array of 4x4 sensors, 16 in vitro tissues are formed, each in separate wells (Fig. 3A). The setup is placed under the microscope and the tissue samples are monitored for 48 hours. This method enables quantification of force using displacement of grips. The specimens are also stretched at different time points to measure stiffness, alteration of which gives an indication towards remodeling of ECM due to cellular activities.

Results, Conclusions, and Discussions: Using the novel high-throughput sensor system, we investigated the effects of chemotherapy drugs on human colorectal cancer associated fibroblasts (CAFs), human pancreatic stellate cells (PSCs) and patient-derived pancreatic ductal adenocarcinoma (PDAC) organoids. In particular, we examined how their contractility and matrix remodeling capacity was affected by different drug combinations and dosages. These results, in our opinion, provide the first evidence of mechanobiology based assessment of drug efficacy in 3D tumor models.
Our results with CAFs and PSCs challenged with investigational drug ATRA (all-trans retinoic acid) show that ATRA can possibly reverse matrix stiffening by the CAFs. Typically, CAFs increase stiffness of collagen scaffolds (2 mg/ml) by 2-3 folds within 24 hours. After initial stiffening (Fig. 3B), we applied ATRA at concentrations of 1, 2 and 5 uM for the next 24 hours. Surprisingly, treatment with ATRA resulted in softening of the matrices, in contrast with control samples that retained their stiffness (Fig. 3B). Specimens prepared with PSCs within Collagen (2 mg/ml) + Matrigel (1 mg/ml) matrices exhibited similar effects (Fig. 3C). Our hypothesis is that ATRA inhibits contractility of stromal fibroblasts (e.g. CAFs, PSCs) and induces secretion of proteolytic enzymes (e.g. MMPs) that degrade the structure of collagen. However, further investigation is necessary to test this hypothesis.
Next, we examined whether PDAC cell secreted factors increase contractility and matrix stiffening by PSCs. To this end, we applied cancer conditioned media (CM) from human PDAC cell line (Mia PaCa-2) to specimens (PSCs+collagen I+Matrigel) on the sensor array. Interestingly, CM from cancer cells immediately stimulated the PSCs as demonstrated by their rapid increase of contractility and excessive stiffening of the matrices (Fig. 3D).
Finally, to demonstrate translational capabilities of the system, we created in vitro tumor models with patient-derived organoids (PDOs) and PSCs; applied Gemcitiabine (2 uM), ATRA (3.3 uM) and a combination of these; and tracked their contractility and stiffness with time (Fig. 3E-G). We found that the combination of Gemcitabine and ATRA is very efficient in inhibiting force (Fig. 3G) and matrix stiffening (Fig. 3E). These results suggest that the high-throughput system is ready for clinical applications.

Acknowledgements (Optional):

References:
[1] B. Emon, Z. Li, M.S.H. Joy, U. Doha, F. Kosari, M.T.A. Saif, A novel method for sensor-based quantification of single/multicellular force dynamics and stiffening in 3D matrices, Sci Adv 7 (2021) eabf2629. https://doi.org/10.1126/sciadv.abf2629.