Cellular and Molecular Bioengineering

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.\n\nSynopsisCryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors.

Controlling basic fibroblast growth factor (bFGF) signaling is important for both tissue-engineering purposes, controlling proliferation and differentiation potential, and for cancer biology, influencing tumor progression and metastasis. Here, we observed that human mesenchymal stromal cells (hMSCs) no longer responded to soluble or covalently bound bFGF when cultured on microfibrillar substrates, while fibroblasts did. This correlated with a downregulation of FGF receptor 1 (FGFR1) expression of hMSCs on microfibrillar substrates, compared to hMSCs on conventional tissue culture plastic (TCP). hMSCs also expressed less SRF on ESP scaffolds, compared to TCP, while fibroblasts maintained high FGFR1 and SRF expression. Inhibition of actin-myosin tension or the MRTF/SRF pathway decreased FGFR1 expression in hMSCs, fibroblasts and MG63 osteosarcoma cells. This downregulation was functional, as hMSCs became irresponsive to bFGF in the presence of MRTF/SRF inhibitor. Together, our data show that hMSCs, but not fibroblasts, are irresponsive to bFGF when cultured on microfibrillar susbtrates by downregulation of FGFR1 through the MRTF/SRF pathway. This is the first time FGFR1 expression has been shown to be mechanosensitive and adds to the sparse literature on FGFR1 regulation. These results could open up new targets for cancer treatments and could aid designing tissue engineering constructs that better control cell proliferation.

Systemic chemotherapeutics target cancer cells but are also known to impact other cells away from the tumor. Questions remain whether systemic chemotherapy crosses the blood-brain barrier and causes inflammation in the periphery that impacts the central nervous system (CNS) downstream. The meningeal lymphatics are a critical component that drain cerebrospinal fluid from the CNS to the cervical lymph nodes for immunosurveillence. To develop new tools for understanding chemotherapy-mediated effects on the meningeal lymphatics, we present two novel models that examine cellular and tissue level changes. Our in vitro tissue engineered model of a meningeal lymphatic vessel lumen, using a simple tissue culture insert system with both lymphatic endothelial and meningeal cells, examines cell disruption. Our ex vivo model culturing mouse meningeal layers probes structural changes and remodeling, correlating to an explant tissue level. To gain a holistic understanding, we compare our in vitro and ex vivo models to in vivo studies for validation and a three-tier methodology for examining the chemotherapeutic response of the meningeal lymphatics. We have demonstrated that the meningeal lymphatics can be disrupted by systemic chemotherapy but show differential responses to platinum and taxane chemotherapies, emphasizing the need for further study of off-target impacts in the CNS.

Epithelial cells are well-known to be modulated by extracellular mechanical factors including substrate stiffness. However, the effect of substrate stiffness on an epithelial cells principal function -creating an effective barrier to protect the underlying tissue - cannot be directly measured using existing experimental techniques. We developed a strategy involving ethylenediamine aminolysis and glutaraldehyde crosslinking to chemically graft polyacrylamide hydrogels with tunable stiffness to PET Transwell membranes. Grafting success was evaluated using spectroscopic methods, scrape tests, and extended incubation in culture. By assessing apical to basolateral transfer of fluorescent tracers, we demonstrated that our model is permeable to biologically relevant molecules and usable for direct measurement of barrier function by calculating paracellular permeability.\n\nWe found that BEAS-2B epithelial cells form a more effective barrier on stiff substrates, likely attributable to increased cell spreading. We also observed barrier impairment after treatment with transforming growth factor beta, indicating loss of cell-cell junctions, validating our models ability to detect biologically relevant stimuli. Thus, we have created an experimental model that allows explicit measurement of epithelial barrier function for cells grown on different substrate stiffnesses. This model will be a valuable tool to study mechanical regulation of epithelial and endothelial barrier function in health and disease.

Drug resistance remains a major challenge in cancer treatment by contributing to recurrence and metastasis. Fractional killing, in which only a subset of cells undergo apoptosis after drug exposure, is a key contributor to this resistance and is influenced by genetic and nongenetic heterogeneity within the tumor microenvironment. Solid tumors display substantial variation in extracellular matrix stiffness, providing evidence that the mechanical context of cancer and stromal cells may play an important role in therapeutic response. Here, we investigated how substrate stiffness affects the dynamics of apoptosis and the mechanisms behind differences in the cell death response to doxorubicin (DOX). HeLa cells cultured on stiffer substrates exhibited enhanced caspase-3/7 activation and increased apoptotic cell death, whereas cells on soft substrates showed markedly reduced apoptotic signaling and improved survival. Although substrate stiffness altered cytoskeletal organization, pharmacological disruption of actin polymerization or actomyosin contractility did not influence nuclear DOX accumulation, indicating that cytoskeletal mechanics were not the primary factor in the stiffness-dependent sensitivity. Instead, flow cytometry revealed that substrate stiffness modulates cell-cycle distribution, with soft substrates enriched in the G1 population and a reduced fraction of cells in the DOX-sensitive S phase. Synchronizing cells at the G1/S phase boundary eliminated stiffness-dependent differences in apoptotic activation, demonstrating that cell-cycle state is a dominant driver of stiffness-mediated fractional killing. These findings highlight a mechanistic link between extracellular matrix mechanics and chemotherapeutic response by suggesting that microenvironment-regulated cell-cycle dynamics contribute to drug resistance in mechanically heterogeneous tumors.

Cells sense the stiffness of their extracellular matrix (ECM) and adapt their behavior accordingly. We investigated how ECM stiffness affects the spatial organization of talin1, a key mechanosensitive focal adhesion protein. Using polyacrylamide (PA) hydrogels with tunable stiffnesses (0.2-188 kPa), we analyzed cell morphology, migration, talin1 distribution, colocalization with tensin3, and fibronectin deposition. Softer substrates enhanced filopodia activity and altered migration behavior. On softer ECMs, talin1 displayed a more even intracellular distribution, whereas on stiffer matrices it localized to the cell periphery. PA gels supported elongated talin1-based adhesions, whose morphology showed minimal variation across the 3-188 kPa stiffness range. Talin1-tensin3 colocalization was maintained regardless of stiffness, indicating a stable interaction. Notably, cells deposited more fibronectin on softer substrates. While talin1 adhesion morphology varied little with stiffness, cell migration behavior changed markedly. Combined with prior studies, our data suggests that ECM stiffness regulates talin1 primarily through conformational changes rather than macroscopic adhesion remodeling. These findings highlight talin1s central role in translating mechanical cues into dynamic cellular responses. Summary statementTalin1 forms elongated adhesions and robustly colocalizes with tensin3 across varying matrix stiffnesses, showing that their spatial organization is largely insensitive to mechanical cues.

Glioblastoma (GBM) is one of the most common malignant brain tumors, with patient mortality driven by invasion into the surround brain microenvironment and drug resistance. Multicellular spheroids are increasingly a common model to study GBM invasion and drug response in engineered biomaterials. However, a key design feature of tumor spheroid studies is the size of each spheroid (number of cells, diameter). Given the heterogenous growth of GBM cells at the surgical margin, spheroids of different sizes may also have clinical relevance. Here, we define shifts in behavior and drug response of wild type and temozolomide (TMZ) resistant GBM spheroids as a function of initial spheroid size. GBM spheroids ranging from 100 to 12,000 cells in size were embedded into a methacrylamide-functionalized gelatin (GelMA) hydrogel. GBM spheroid size had an inverse relationship with the number of apoptotic cells. We observed significant spheroid size dependent effects on TMZ efficacy for both TMZ resistant and wild type cells. Interestingly, high single doses of TMZ were more effective in reducing three-dimensional migration from smaller spheroids than metronomic dosing while high single dose and metronomic dosing were equally effective in reducing invasion for large TMZ-resistant spheroids. Our study highlights the importance of considering and reporting spheroid size for cancer tissue engineering studies considering invasion and drug resistance. It also informs future studies of residual GBM at the tumor margins most responsible for patient relapse and mortality.

Biomaterials, like polydimethylsiloxane (PDMS), are soft, biocompatible, and tuneable, which makes them useful to delineate specific substrate factors that regulate the complex landscape of cell-substrate interactions. We used a commercial formulation of PDMS to fabricate substrates with moduli 40 kPa, 300 kPa, and 1.5 MPa, and cultured HMF3S fibroblasts on them. Gene expression analysis was performed by RT-PCR and Western blotting. Cellular and nuclear morphologies were analyzed using confocal imaging, and MMP-2 and MMP-9 activities were determined with gelatin zymography. Results, comparing mechanotransduction on PDMS substrates with control petridishes, show that substrate stiffness modulates cell morphologies and proliferations. Cell nuclei were rounded on compliant substrates and correlated with increased tubulin expression. Proliferations were higher on stiffer substrates with cell cycle arrest on softer substrates. Integrin 5 expression decreased on lower stiffness substrates, and correlated with inefficient TGF-{beta} activation. Changes to the activated state of the fibroblast on higher stiffness substrates were linked to altered TGF-{beta} secretion. Collagen I, collagen III, and MMP-2 expression levels were lower on compliant PDMS substrates as compared to stiffer ones; there was little MMP-9 activity on substrates. These results demonstrate critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts which is highly relevant in diseases like tissue fibrosis.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related deaths in the U.S., despite only being the 11th most common cancer. The high mortality rates of PDAC can be partially attributed to the tumor microenvironment. Unlike most carcinomas, PDAC is characterized by a strong desmoplastic reaction, or a fibrotic stiffening of the extracellular matrix (ECM) in response to chronic inflammation. The desmoplastic reaction is mediated by cancer-associated fibroblasts that deposit ECM proteins (collagens, laminins, fibronectin, etc.) and secrete matrix-remodeling proteins in the tumor parenchyma. Within the past decade, the enzyme quiescin sulfhydryl oxidase 1 (QSOX1) has gained recognition as a significant contributor to solid tumor pathogenesis, but its biological role remains uncertain. QSOX1 is a disulfide bond-generating catalyst that participates in oxidative protein folding in the mammalian cell. Current studies show that inhibiting or knocking down QSOX1 reduces pancreatic cancer cell migration and invasion, alters ECM deposition and organization, and decreases overall tumor growth in mice. However, it is unclear which features of the tumor microenvironment modulate QSOX1 and cause its overexpression in cancer. In this study, we explored potential regulators of QSOX1 expression and secretion by testing two major features of PDAC: hypoxia and mechanical stiffness. To induce hypoxia, we exposed pancreatic cancer cells to atmospheric (low O2) and chemical (CoCl2) hypoxia for up to 48 hours. QSOX1 gene and protein expression did not change in response to hypoxia. Substratum stiffness was modulated using polyacrylamide gels to represent the dynamic pathological range of elastic moduli found in PDAC tissue. We discovered that QSOX1 levels were decreased on softer surfaces compared to conventional tissue culture plastic. This paper presents new results and challenges prior findings on QSOX1 regulation in pancreatic tumor cells.

Monocytes are members of the mononuclear phagocyte system involved in pathogen clearance and nanoparticle pharmacokinetics. Monocytes play a critical role in the development and progression of cardiovascular disease and, recently, in SARS-CoV-2 pathogenesis. While studies have investigated the effect of nanoparticle modulation on monocyte uptake, their capacity for nanoparticle clearance is poorly studied. In this study, we investigated the impact of ACE2 deficiency, frequently observed in individuals with cardiovascular complications, on monocyte nanoparticle endocytosis. Moreover, we investigated nanoparticle uptake as a function of nanoparticle size, physiological shear stress, and monocyte phenotype. Our Design of Experiment (DOE) analysis found that the THP-1 ACE2- cells showed a greater preference for 100nm particles under atherosclerotic conditions than THP-1 wild-type cells. Observing how nanoparticles can modulate monocytes in the context of disease can inform precision dosing.

Glioblastoma (GBM) tumor cells are found in the perivascular niche microenvironment and are believed to associate closely with the brain microvasculature. However, it is largely unknown how the resident cells of the perivascular niche, such as endothelial cells, pericytes, and astrocytes, influence GBM tumor cell behavior and disease progression. We describe a three-dimensional in vitro model of the brain perivascular niche developed by encapsulating brain-derived endothelial cells, pericytes, and astrocytes in a gelatin hydrogel. We show that pericytes and astrocytes explicitly contribute to vascular architecture and maturation. We use co-cultures of patient-derived GBM tumor cells with brain microvascular cells to identify a role for pericytes and astrocytes in establishing a perivascular niche environment that modulates GBM cell invasion, proliferation, and therapeutic response. Engineered models provides unique insight regarding the spatial patterning of GBM cell phenotypes in response to a multicellular model of the perivascular niche. Critically, we show that engineered perivascular models provide an important resource to evaluate mechanisms by which inter- cellular interactions modulate GBM tumor cell behavior, drug response, and provide a framework to consider patient-specific disease phenotypes.

Polymeric hydrogels are valuable platforms for determining how specific mechanical properties of native tissue extracellular matrix (ECM) regulate cell function. Recent research has focused on incorporating viscous and elastic properties into hydrogels to investigate cellular responses to time-dependent mechanical properties of the ECM. However, a critical aspect often overlooked is that cells continuously remodel their microenvironment in hydrogels, such as by the deposition of newly secreted (nascent) ECM. While this nascent ECM has been demonstrated to play a vital role in transmitting mechanical signals across various biological contexts, the mechanisms by which it regulates cellular function in response to time-dependent mechanical properties remain poorly understood. In this study, we developed an interpenetrating polymer network that enables independent control of viscous and elastic hydrogel properties. We show that cells cultured on high-viscosity hydrogels deposit increased nascent ECM which also correlates with enhanced hydrogel remodeling. Interestingly, higher nascent ECM deposition on high-viscosity hydrogels was decoupled from intracellular contractility. These results establish a relationship between hydrogel viscosity and nascent ECM deposition that may extend to diverse cell types and offer new insights into cell-hydrogel interactions.

Epithelial cell migration is critical in regulating wound healing and tissue development. The epithelial microenvironment is incredibly dynamic, subjected to mechanical cues including cyclic stretch. While cyclic cell stretching platforms have revealed responses of the epithelium such as cell reorientation and gap formation, few studies have investigated the long-term effects of cyclic stretch on cell migration. We measured the migratory response of the epithelium to a range of physiologically relevant frequencies and stretch. We integrated our experimental approach with high-throughput cell segmentation to discover a relationship between changes in cell morphology and migration as a function of cyclic stretch. Our results indicate that lower stretch frequencies (i.e., 0.1 Hz) arrest epithelial migration, accompanied by cell reorientation and high cell shape solidity. We found that this response is also accompanied by increased recruitment of vinculin to cell-cell contacts, and this recruitment is necessary to arrest cell movements. This work demonstrates a critical role for frequency dependence in epithelial response to mechanical stretch. These results confirm the mechanosensitive nature of vinculin within the adherens junction, but independently reveal a novel mechanism of low frequency stress response in supporting epithelial integrity by arresting cell migration.

Trophoblast invasion is a complex biological process necessary for establishment of pregnancy; however, much remains unknown regarding what signaling factors coordinate the extent of invasion. Pregnancy-specific glycoproteins (PSGs) are some of the most abundant circulating trophoblastic proteins in maternal blood during human pregnancy, with maternal serum concentrations rising to as high as 200-400 g/mL at term. Here, we employ three-dimensional (3D) trophoblast motility assays consisting of trophoblast spheroids encapsulated in 3D gelatin hydrogels to quantify trophoblast outgrowth area, viability, and cytotoxicity in the presence of PSG1 and PSG9 as well as epidermal growth factor and Nodal. We show PSG9 reduces trophoblast motility whereas PSG1 increases motility. Further, we assess bulk nascent protein production by encapsulated spheroids to highlight the potential of this approach to assess trophoblast response (motility, remodeling) to soluble factors and extracellular matrix cues. Such models provide an important platform to develop a deeper understanding of early pregnancy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/314195v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@9062aaorg.highwire.dtl.DTLVardef@13dbc87org.highwire.dtl.DTLVardef@2473eaorg.highwire.dtl.DTLVardef@1362fba_HPS_FORMAT_FIGEXP M_FIG C_FIG

Carcinomas commonly recur and progress rapidly after a period of remission following platinum-based therapy. This clinical scenario suggests that surviving drug-resistant tumor cells are dormant or slow cycling before re-entering a rapid growth phase. Remodeling of the recurrent tumor microenvironment (TME) contributes to high rates of metastasis, but little is known about differences in TME remodeling before therapy and after recurrence. This study explores selection for cisplatin-resistant subpopulations of A549 lung adenocarcinoma cells in culture to derive populations for modeling features of the recurrent TME. A cisplatin dose of 25 M killed approximately 80% of the cells while sparing enough cells to allow re-expansion of sufficient cell numbers for downstream experimentation. Expanded cisplatin-resistant derivatives (Cis-R A549) exhibited features of mesenchymal transition (EMT) such as cellular hypertrophy, loss of cell-cell contacts, and upregulation of alpha smooth muscle actin mRNA. In 3D culture, Cis-R A549 spheroids were loosely aggregated and dysmorphic in comparison to the compact and spherical parent A549 spheroids. The Ki67 index of Cis-R A549 in 2D and 3D spheroid culture was markedly lower than parent A549, suggesting a state of pseudo-dormancy with slow cycling. Cis-R A549 upregulated multiple genes associated with the evolution of a more aggressive TME and displayed significantly increased proangiogenic capacity in a microphysiological model of tumor angiogenesis. This study establishes a methodological framework for engineering the recurrent TME with drug-resistant cancer cell line derivatives selected via high-dose exposure in culture. Increased angiogenesis induced by Cis-R A549 suggests that anti-angiogenic therapy may be more beneficial in the setting of recurrent disease following first-line therapies.

Cytotoxic T lymphocytes (CTLs) are important in controlling some viral infections, and therapies involving transfer of large numbers of cancer-specific CTLs have been successfully used to treat several types of cancers in humans. While molecular mechanisms of how CTLs kill their targets are relatively well understood we still lack solid quantitative understanding of the kinetics and efficiency at which CTLs kill their targets in different conditions. Collagen-fibrin gel-based assays provide a tissue-like environment for the migration of CTLs, making them an attractive system to study the cytotoxicity in vitro. Budhu et al. [1] systematically varied the number of peptide (SIINFEKL)- pulsed B16 melanoma cells and SIINFEKL-specific CTLs (OT-1) and measured remaining targets at different times after target and CTL co-inoculation into collagen-fibrin gels. The authors proposed that their data were consistent with a simple model in which tumors grow exponentially and are killed by CTLs at a per capita rate proportional to the CTL density in the gel. By fitting several alternative mathematical models to these data we found that this simple "exponential-growth-mass-action-killing" model does not precisely fit the data. However, determining the best fit model proved difficult because the best performing model was dependent on the specific dataset chosen for the analysis. When considering all data that include biologically realistic CTL concentrations (E [≤] 107 cell/ml) the model in which tumors grow exponentially and CTLs suppress tumors growth non-lytically and kill tumors according to the mass-action law (SiGMA model) fitted the data with best quality. Results of power analysis suggested that longer experiments ([~] 3 - 4 days) with 4 measurements of B16 tumor cell concentrations for a range of CTL concentrations would best allow to discriminate between alternative models. Taken together, our results suggest that interactions between tumors and CTLs in collagen-fibrin gels are more complex than a simple exponential-growth- mass-action killing model and provide support for the hypothesis that CTLs impact on tumors may go beyond direct cytotoxicity.

Cancer researchers now consider spheroids a valuable in vitro model for cancer research and personalized medicine. They are used to studying cancer development, testing drug effectiveness, and potentially guiding treatment decisions for individual patients. Spheroids represent the simplest form of three-dimensional (3D) cellular arrangement and encapsulate the essential tumor microenvironment. These characteristics are crucial for studying processes such as tumor invasion, metastasis, angiogenesis, and cell cycle kinetics. In particular, spheroids excel in chemo-response assays where traditional monolayer cell cultures often fall short. A PDMS microwell device was developed to generate uniform-sized cancer spheroids. This device is user-friendly and capable of producing a large number of spheroids. The device measures 13 mm in diameter (1200 microwells per well if the device has microwells 400 m in size, and 300 microwells per well if the device has microwells 800 m in size). It advances 3D cultures by requiring only a small volume of cell culture supplements and is easy to manage. The hydrophobic nature of the PDMS device prevents cells from adhering to it, thereby promoting spheroid formation. Spheroids can be created on microwell devices, and subsequent experiments may either be conducted on the device or transferred to cell culture dishes for additional 3D biological assays. Seeding cells is notably easier compared to other 3D cell culture techniques, and the number of cells in each spheroid can be adjusted according to specific requirements. Overall, the PDMS-based microwell device offers a simple and efficient means to produce large quantities of uniform-sized spheroids for 3D cell culture studies, showcasing high throughput, short generation times, long-term effectiveness, and ease of handling.

Brain cells are influenced by continuous fluid shear stress driven by varying hydrostatic and osmotic pressure conditions, depending on the brains pathophysiological conditions. While all brain cells are sensitive to the subtle changes in various physicochemical factors in the microenvironment, microglia, the resident brain immune cells, exhibit the most dramatic morphodynamic transformation. However, little is known about the phenotypic alterations in microglia in response to the changes in fluid shear stress. In this study, we first established a flow-controlled microenvironment to investigate the effects of shear flow on microglial phenotypes, including morphology, motility, and activation states. Microglia exhibited two distinct morphologies with different migratory phenotypes in a static condition: bipolar cells that oscillate along their long axis and unipolar cells that migrate persistently. When exposed to flow, a significant fraction of bipolar cells showed unstable oscillation with an increased amplitude of oscillation and a decreased frequency, which consequently led to the phenotypic transformation of oscillating cells into migrating cells. Interestingly, the level of pro-inflammatory genes increased in response to shear stress, while there were no significant changes in the level of anti-inflammatory genes. Our findings suggest that an interstitial fluid-level stimulus can cause a dramatic phenotypic shift in microglia toward pro-inflammatory states, shedding light on pathological outbreaks of severe brain diseases. Given that the fluidic environment in the brain can be locally disrupted in pathological circumstances, the mechanical stimulus by a fluid flow should also be considered a crucial element in regulating the immune activities of the microglia in brain diseases. Statement of SignificanceCellular morphology and motility are important factors that encompass the alterations in protein and gene-level expressions within cells. In pathological conditions, microglia, the resident brain immune cells, are known to undergo morphodynamic transformations in response to various physicochemical stimuli. Besides the commonly known soluble biochemical factors in the microenvironment, the differential flow characteristics of ISF have been linked to several neurological diseases, such as Alzheimers, Parkinsons, and brain tumors. Microglial cells, which are extremely sensitive to subtle changes in extracellular stimuli, have been identified as key players in these pathological conditions. Despite its importance, however, it has been challenging to study the sole effect of a shear flow on microglia. We investigated the morphodynamic features of microglia in response to precisely controlled interstitial-level fluid flow conditions using a microfluidic system in which isolated microglia are monitored in real-time while the undesirable effects from other extracellular factors are minimized.

Antibody-drug conjugates (ADCs) have had remarkable clinical success in recent years with multiple new approvals. However, for some ADCs, the response rates dont closely correlate with clinical target expression. One particular ADC targeting HER2, trastuzumab deruxtecan or T-DXd, is notable due to its success at expression levels ranging from high to low and ultralow. This raises the question of the relative contributions of target-independent mechanisms on ADC efficacy in the clinic, and several such mechanisms have been proposed. However, in vitro and preclinical data have different doses and exposures, making it challenging to quantitatively extrapolate preclinical data to the clinic. In this work, we use our computational hybrid agent-based model, SimADC, to simulate target-dependent and -independent mechanisms, scaling from mice to humans. We first demonstrate that CD8+ T cells can significantly contribute to tumor regression, especially when the ADC further activates the immune cells. Next, we test target-independent payload-driven mechanisms including: 1) Fc-mediated internalization of ADC by intratumoral macrophages and payload release to neighboring cancer cells, 2) free payload circulating in the blood and re-entering the tumor, and 3) extracellular linker cleavage and payload release due to an abundance of proteases in the tumor. We find that free payload in the blood and extracellular linker cleavage had low and moderate impacts, respectively, while macrophage uptake and payload release resulted in high levels of efficacy. This is due to the macrophages ability to sustain free payload in the tumor. Moderate and high HER2 expression were more efficacious than target-independent mechanisms. Overall, our simulations demonstrate that moderate to high HER2 expression, immune activation, or macrophage uptake and payload release are sufficient for T-DXd tumor regression. Additionally, SimADC provides a robust framework for modeling both target-dependent and target-independent mechanisms for any ADC, providing the opportunity to engineer more effective therapeutic agents. Author SummaryCancer is one of the most prevalent diseases in the world, impacting the lives of millions of people every year. Antibody-drug conjugates (ADCs) are a form of targeted therapy that can deliver cytotoxic drugs directly to cancer cells, increasing efficacy. However, ADCs are complex to design and test, as each part of the ADC (targeting antibody, cytotoxic payload, and linker) must be optimally selected for delivery for each target and type of patient. Here, we studied ADCs using a computational model, which allowed us to simulate ADCs in varying cancer environments efficiently and economically. We validated our model using preclinical data to incorporate patient immune responses, target-independent payload release, and systemic payload uptake, allowing us to make accurate predictions in mice and extrapolate to human tumors. We compared multiple mechanisms by which ADCs can kill cancer cells to help identify the most effective methods. Besides high target expression, immune stimulation and target-independent release in the microenvironment can contribute to tumor regression. Investigating these mechanisms enables the design of ADCs and treatment regimens that maximize efficacy across a range of tumor types and target expression.

The tumor microenvironment (TME), which is composed of various cell types and the extracellular matrix (ECM), plays crucial roles in cancer progression and treatment outcomes. However, the impact of the mechanical properties of the ECM, specifically collagen fibril alignment and crosslinking, on macrophage behavior and polarization is less understood. To investigate this, we reconstituted 3D collagen matrices to mimic the physical characteristics of the TME. Our results demonstrated that stiffening the matrix through the alignment or crosslinking of collagen fibrils promotes macrophage polarization toward the anti-inflammatory M2 phenotype. This phenotype is characterized by increased expression of CD105 and CD206 and a distinct cytokine secretion profile. The increased stiffness and aligned fibrils activate mechanotransduction pathways, notably integrin {beta}1 and PI3K signaling, leading to increased IL-4 secretion, which acts in an autocrine manner to further promote M2 polarization. Interestingly, these stiffened microenvironments also suppressed the proinflammatory response. In coculture experiments with breast cancer cell lines (MDA-MB-231 and MCF-7), macrophages within stiffened or aligned matrices significantly increased cancer cell proliferation and invasion. These findings suggest that the mechanical properties of the ECM, specifically its alignment and crosslinking, create a more favorable environment for tumor progression by modulating macrophage activity. Overall, our study underscores the critical role of ECM mechanics in shaping immune cell behavior within the TME, highlighting the potential for therapies that target ECM properties and macrophage polarization to inhibit cancer progression and enhance treatment efficacy.