Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly elucidated. Here, comparative transcriptomic analyses of mouse LNSC subsets demonstrated the expression of important immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate receptors across hematopoietic and stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response and the antigen-processing and antigen-presentation machinery. Poorly studied podoplanin (gp38)-negative CD31(-) LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified as myofibroblastic pericytes that expressed integrin α(7). Together our data comprehensively describe the transcriptional characteristics of LNSC subsets.

Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes.

Fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) are nonhematopoietic stromal cells of lymphoid organs. They influence the migration and homeostasis of naive T cells; however, their influence on activated T cells remains undescribed. Here we report that FRCs and LECs inhibited T cell proliferation through a tightly regulated mechanism dependent on nitric oxide synthase 2 (NOS2). Expression of NOS2 and production of nitric oxide paralleled the activation of T cells and required a tripartite synergism of interferon-γ, tumor necrosis factor and direct contact with activated T cells. Notably, in vivo expression of NOS2 by FRCs and LECs regulated the size of the activated T cell pool. Our study elucidates an as-yet-unrecognized role for the lymph node stromal niche in controlling T cell responses.

Novel combination of bioactive agents in bilayered dermal patches provides superior wound healing.

In present study, multifunctional bilayered dermal patches with antibacterial, antioxidant and anti-inflammatory properties were developed using solvent casting or electrospinning methods and compared for performance. Top layer was made up of polycaprolactone (PCL) and chitosan (CS) while bottom layer was made of polyvinyl alcohol (PVA) with curcumin nanoparticles and soluble eggshell membrane protein (SESM) as the wound healing agents. Curcumin nanoparticles showed reduction in the production of reactive oxygen species (ROS) and inflammatory cytokines and markers in mice T cells or human macrophages, confirming their antioxidant and anti-inflammatory properties while SESM improved migration of human adult dermal fibroblasts, suggesting its contribution to wound healing. The dermal patches were hemocompatible and antibacterial and also provided adequate absorption of wound exudates, support and components required for recruitment of cells and deposition of extracellular matrix to enable superior wound healing than its commercial counterpart in a full thickness excision wound model in rats.

Differential Influence of IL-9 and IL-17 on Actin Cytoskeleton Regulates the Migration Potential of Human Keratinocytes.

T cells mediate skin immune surveillance by secreting specific cytokines and regulate numerous functions of keratinocytes, including migration during homeostasis and disease pathogenesis. Keratinocyte migration is mediated mainly by proteolytic cleavage of the extracellular matrix and/or by cytoskeleton reorganization. However, the cross-talk between T cell cytokines and actomyosin machinery of human primary keratinocytes (HPKs), which is required for cytoskeleton reorganization and subsequent migration, remains poorly examined. In this study, we describe that IL-9 profoundly reduced the actin stress fibers, inhibited contractility, and reduced the cortical stiffness of HPKs, which resulted in inhibition of the migration potential of HPKs in an adhesion- and MMP-independent manner. Similarly, IL-9 inhibited the IFN-γ-induced migration of HPKs by inhibiting the actomyosin machinery (actin stress fibers, contractility, and stiffness). IL-17A increased the actin stress fibers, promoted cellular contractility, and increased proteolytic collagen degradation, resulting in increased migration potential of HPKs. However, IL-9 inhibited the IL-17A-mediated HPKs migration. Mechanistically, IL-9 inhibited the IFN-γ- and IL-17A-induced phosphorylation of myosin L chain in HPKs, which is a major regulator of the actomyosin cytoskeleton. Finally, in addition to HPKs, IL-9 inhibited the migration of A-431 cells (epidermoid carcinoma cells) induced either by IFN-γ or IL-17A. In conclusion, our data demonstrate the influence of T cell cytokines in differentially regulating the actomyosin cytoskeleton and migration potential of human keratinocytes, which may have critical roles in skin homeostasis and pathogenesis of inflammatory diseases as well as skin malignancies.

Gelatin Methacryloyl Based Injectable Cryogels with Tunable Degradability for Cell Delivery.

Scaffold-based cell delivery can improve therapeutic effects of transplanted cells in cell therapy. Biomaterial scaffolds serveas niche for cell growth and proliferation which improves cell survival and overall function post cell delivery. In this study, gelatin methacryloyl based injectable scaffolds made using poly(ethylene)glycol as a sacrificial polymer and cryogelation as a technique, are demonstrated to have tunable degradability and porosity that is required for cell and drug delivery applications. The pore size (10-142 µm) of these gels makes them suitable for loading different cell types as per the application. In vitro studies using mammalian cells confirm that these cryogels are cytocompatible. These cell-laden scaffolds are injectable and have a cell retention ability of up to 90% after injection. Rheology is done to evaluate stiffness and shape recovery property, and it is found that these gels can maintain their original shape even after applying 7 cycles of strain from 0.1% to 20%. Furthermore, their degradability can be modulated between 6 and 10 days by changing the overall polymer composition. Thus, injectability and degradability of these cryogels can circumvent invasive surgical procedures, thereby making them useful for a variety of applications including delivery of cells and bioactive factors.

Surface-modified injectable poly(ethylene-glycol) diacrylate-based cryogels for localized gene delivery.

Lentiviral transduction is widely used in research, has shown promise in clinical trials involving gene therapy and has been approved for CAR-T cell immunotherapy. However, most modifications are doneex vivoand rely on systemic administration of large numbers of transduced cells for clinical applications. A novel approach utilizingin situbiomaterial-based gene delivery can reduce off-target side effects while enhancing effectiveness of the manipulation process. In this study, poly(ethylene glycol) diacrylate (PEGDA)-based scaffolds were developed to enablein situlentivirus-mediated transduction. Compared to other widely popular biomaterials, PEGDA stands out due to its robustness and cost-effectiveness. These scaffolds, prepared via cryogelation, are capable of flowing through surgical needles in bothin vitroandin vivoconditions, and promptly regain their original shape. Modification with poly(L-lysine) (PLL) enables lentivirus immobilization while interconnected macroporous structure allows cell infiltration into these matrices, thereby facilitating cell-virus interaction over a large surface area for efficient transduction. Notably, these preformed injectable scaffolds demonstrate hemocompatibility, cell viability and minimally inflammatory response as shown by ourin vitroandin vivostudies involving histology and immunophenotyping of infiltrating cells. This study marks the first instance of using preformed injectable scaffolds for delivery of lentivectors, which offers a non-invasive and localized approach for delivery of factors enablingin situlentiviral transduction suitable for both tissue engineering and immunotherapeutic applications.

Acellular scaffold-based approach for in situ genetic engineering of host T-cells in solid tumor immunotherapy.

BACKGROUND: Targeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies. However, its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity. Localized delivery of tumor-specific T-cells using biomaterials has shown promise, however, procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints. METHODS: Polyethylene glycol (PEG)-based three-dimensional (3D) scaffolds were developed and conjugated with positively charged poly-L-lysine (PLL) using carbamide chemistry for efficient loading of lentiviruses (LVs) carrying tumor antigen-specific T-cell receptors (TCRs). The physical and biological properties of the scaffold were extensively characterized. Further, the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin (B16-OVA) tumor model to evaluate the anti-tumor response and the presence of transduced T-cells. RESULTS: Our findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site. In B16-OVA melanoma tumor-bearing mice, the scaffolds efficiently transduce host T-cells with OVA-specific TCRs. These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs, resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines. Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds. CONCLUSION: Our scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors. This approach offers a viable alternative to in vitro manipulation of T-cells, circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells. It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells, thereby, overcoming a major hurdle.

Inflammatory cytokines presented from polymer matrices differentially generate and activate DCs in situ.

During infection, inflammatory cytokines mobilize and activate dendritic cells (DCs), which are essential for efficacious T cell priming and immune responses that clear the infection. Here we designed macroporous poly(lactide-co-glycolide) (PLG) matrices to release the inflammatory cytokines GM-CSF, Flt3L and CCL20, in order to mimic infection-induced DC recruitment. We then tested the ability of these infection mimics to function as cancer vaccines via induction of specific, anti-tumor T cell responses. All vaccine systems tested were able to confer specific anti-tumor T cell responses and longterm survival in a therapeutic, B16-F10 melanoma model. However, GM-CSF and Flt3L vaccines resulted in similar survival rates, and outperformed CCL20 loaded scaffolds, even though they had differential effects on DC recruitment and generation. GM-CSF signaling was identified as the most potent chemotactic factor for conventional DCs and significantly enhanced surface expression of MHC(II) and CD86(+), which are utilized for priming T cell immunity. In contrast, Flt3L vaccines led to greater numbers of plasmacytoid DCs (pDCs), correlating with increased levels of T cell priming cytokines that amplify T cell responses. These results demonstrate that 3D polymer matrices modified to present inflammatory cytokines may be utilized to effectively mobilize and activate different DC subsets in vivo for immunotherapy.

Choline ester based ionic liquid: A multi-functional system to enhance nucleic acid stability, drug solubilization and cell penetration.

Ionic liquids (ILs) are emerging systems with applications in varying areas of biomedical research. This study aims at developing a biocompatible, dual function choline ester-based IL with chloride as anion ([Ch] IL) for stabilizing nucleic acids (DNA) and enhancing cellular uptake of drugs. The ability of IL to complex with DNA was characterized using electrophoresis, dye displacement and UV absorbance. The effect of pH on complex stability and protection of DNA from nuclease were also studied. Even though [Ch] IL had positive zeta potential and showed effective complex formation, at physiological pH the zeta potential of the complex decreased and became negative, thereby, destabilizing the complex. To address this, citric acid (CA) was added to [Ch] IL which facilitated strong complexation. Further, DNA could be retrieved from these complexes without compromising its purity and integrity. Additionally, [Ch] IL was found to improve the cellular uptake of doxorubicin by improving its solubility in water. Thus, we demonstrate that the [Ch] IL developed here can enhance nucleic acid stability, drug solubilization and cell penetration. Our results show that the developed [Ch] IL can be used for long term storage of nucleic acids as well as for enhancing permeation of drugs in vivo.

An interplay of matrix stiffness, dimensionality and adhesivity on cellular behavior.

Cell-based assays are essentialin vitrotools for understanding basic cell biology, pathophysiology of diseases and mechanism of drug actions most cancer studies have utilized two-dimensional (2D) cell culture methods, which have their shortcomings including lack of cell- extracellular matrix interactions and three-dimensional (3D) geometry, and inaccurate representation of cell polarity. Hence, 3D matrices are being increasingly used to study the effect of 3D niche on cell behavior. Till date, very few systematic studies have been done to show comparison of cell behavior when seeded on the surface and encapsulated inside the matrix. In this study, we fabricated poly(ethylene glycol) (PEG) and gelatin-based matrices using UV mediated photo-polymerization to establish 2D and 3D cell culture methods using breast cancer MDA-MB-231 cells. We have found that the adhesion and spreading of cells on the gel surface is different from that when embedded in gels. The stiffness of poly (ethylene glycol) diacrylate (PEGDA)-gelatin methacryloyl (GelMA) hydrogels with lower concentration of GelMA is lower than that with higher GelMA; further, those with higher overall concentration of polymers (>5%) retain their mechanical integrity and do not degrade even after 7 d. Physical characterization of these matrices demonstrate their optimal pore size, mechanical stiffness and degradation, which are further tunable for tissue engineering, regenerative medicine, drug delivery and cancer studies. Additionally, these semi-synthetic PEGDA-GelMA matrices are transparent in nature, thereby, allowing easy imaging of cells in 3D. The system developed here can be used for short and long term cell culture and can be potentially explored for cell migration and metastasis studies.

Two-photon polymerization based reusable master template to fabricate polymer microneedles for drug delivery.

Microneedle patches have been widely used in a minimally invasive manner for various drug delivery applications. However, for developing these microneedle patches, master molds are required, which are generally made of metal and are very expensive. Two-photon polymerization (2PP) technique can be used for fabricating microneedles more precisely and at a much lower cost. This study reports a novel strategy for developing microneedle master templates using the 2PP method. The main advantage of this technique is that there is no requirement for post-processing after laser writing, and that for the fabrication of polydimethylsiloxane (PDMS) molds, harsh chemical treatments such as silanization are not required. This is a one-step process for manufacturing of microneedle templates which allows easy replication of negative PDMS molds. This is done by adding resin to the master-template and annealing at a specific temperature, thereby making the PDMS peel-off easy and allowing re-use of the master template multiple times. Using this PDMS mold, two types of polyvinyl alcohol (PVA)-rhodamine (RD) microneedle patches were developed, namely, dissolving (D-PVA) and hydrogel (H-PVA) patches and were characterized using suitable techniques. This technique is affordable, efficient and does not require post-processing for development of microneedle templates required for drug delivery applications.•Two photon polymerization can be used for cost-effective fabrication of polymer microneedles for transdermal drug delivery.•Post-processing or surface-modification procedures are not required for these master templates.•Using a simple annealing step, the master template becomes reusable and robust for peeling off polymers like PDMS.

Bilayered skin substitute incorporating rutin nanoparticles for antioxidant, anti-inflammatory, and anti-fibrotic effect.

Hypertrophic scarring in large burns and delayed healing in chronic wounds are consequences of prolonged and aggravated inflammation, sustained infiltration of immune cells, free radical generation, and abundance of inflammatory mediators. Therefore, it is imperative to curb hyperinflammation to expedite wound healing. In this study, rutin nanoparticles (RNPs) were synthesized without an encapsulant and incorporated into eggshell membrane powder-crosslinked gelatin-chitosan cryogels to impart antioxidant and anti-inflammatory properties for treating hyperinflammation. The resultant nanoparticles were found to be 17.53 ± 4.03 nm in size and were stable at room temperature for a month with no visible sedimentation. RNPs were found to be non-cytotoxic and exhibited anti-inflammatory (by increasing IL-10 levels) and antioxidant properties (by controlling the generation of reactive oxygen species and enhancing catalase production in human macrophages). Additionally, RNPs were found to reduce α-SMA expression in fibroblasts, thereby demonstrating their anti-scarring effect. In vivo studies with a bilayered skin substitute constituting an RNP-incorporated cryogel proved that it is biocompatible, does not induce renal toxicity, aids wound healing, and induces better re-epithelialization than the control groups at the initial stages. Thus, RNP-incorporated cryogels containing bilayered skin substitutes are an advanced and novel alternative to commercial dermo-epidermal substitutes that lack anti-inflammatory or anti-scarring properties.

In Vitro 3D Spheroid Model Preserves Tumor Microenvironment of Hot and Cold Breast Cancer Subtypes.

Dynamic interaction of cancer, immune, and stromal cells with extracellular matrix components modulates and resists the response of standard care therapies. To mimic this, an in vitro 3D spheroid model is designed using liquid overlay method to simulate hot (MDA-MB-231) and cold (MCF-7) breast tumor microenvironment (TME). This study shows increased mesenchymal phenotype, stemness, and suppressive microenvironment in MDA-MB-231-spheroids upon exposure to doxorubicin. Intriguingly, the presence of human dermal fibroblasts enhances cancer-associated fibroblast phenotype in MDA-MB-231-spheroids through increased expression of CXCL12 and FSP-1, leading to higher infiltration of immune cells (THP-1 monocytes). However, a suppressive TME is observed in both subtypes, as seen by upregulation of M2-macrophage-specific CD68 and CD206 markers. Specifically, increased PDL-1 expressing tumor-associated macrophages along with FoxP3 expressing T regulatory cells are found in MDA-MB-231-spheroids when cultured with peripheral blood mononuclear cells. Further, it is found that the addition of 1-methyl-tryptophan, a potent indoleamine-2,3-dioxygenase-1 inhibitor, subsides the suppressive phenotype by decreasing the M2 polarization via downregulation of tryptophan metabolism and IL10 expression, particularly in MCF-7 triculture spheroids. Thus, the in vitro 3D spheroid model of TME can be utilized in therapeutics to validate immunomodulatory drugs for various breast cancer subtypes.

Clove Oil-Incorporated Antibacterial Gelatin-Chitosan Cryogels for Tissue Engineering: An In Vitro Study.

Infections are a leading cause of mortality and amputations among patients with burns and chronic wounds, respectively. Moreover, the extensive use of antibiotics has led to the rapid spreading of drug resistance among microorganisms. Alternatively, plant-derived natural products, which have been used as traditional therapies for several centuries, are recently gaining popularity as they are relatively affordable and easily available in many developing countries where modern medications are expensive or unavailable. In this study, clove essential oil is used for its antimicrobial property and is further incorporated into cryogels to increase its bioavailability and prolong its bioactivity. The oil-incorporated cryogels are macroporous, biodegradable, possess mechanical properties similar to commercial skin substitutes, are cytocompatible, antibacterial, and allow long-term sustained release of oil for up to at least 14 days. Additionally, clove oil aids the faster closure of in vitro scratch wounds by improving the migration of fibroblasts. This work presents a novel, bioactive scaffold that has the potential to be used as a dermal substitute and serves as an alternative to commercial skin substitutes.

Bio-Mimicking Brain Vasculature to Investigate the Role of Heterogeneous Shear Stress in Regulating Barrier Integrity.

A continuous, sealed endothelial membrane is essential for the blood-brain barrier (BBB) to protect neurons from toxins present in systemic circulation. Endothelial cells are critical sensors of the capillary environment, where factors like fluid shear stress (FSS) and systemic signaling molecules activate intracellular pathways that either promote or disrupt the BBB. The brain vasculature exhibits complex heterogeneity across the bed, which is challenging to recapitulate in BBB microfluidic models with fixed dimensions and rectangular cross-section microchannels. Here, a Cayley-tree pattern, fabricated using lithography-less, fluid shaping technique in a modified Hele-Shaw cell is used to emulate the brain vasculature in a microfluidic chip. This geometry generates an inherent distribution of heterogeneous FSS, due to smooth variations in branch height and width. hCMEC/D3 endothelial cells cultured in the Cayley-tree designed chip generate a 3D monolayer of brain endothelium with branching hierarchy, enabling the study of the effect of heterogeneous FSS on the brain endothelium. The model is employed to study neuroinflammatory conditions by stimulating the brain endothelium with tumor necrosis factor-α under heterogeneous FSS conditions. The model has immense potential for studies involving drug transport across the BBB, which can be misrepresented in fixed dimension models.

Immunomodulation via macrophages to fight solid tumor malignancies.

The paper 'A C-Terminal Fragment of Adhesion Protein Fibulin-7 Inhibits Growth of Murine Breast Tumor by Regulating Macrophage Reprogramming' by Chakraborty et al. highlights that Fbln7-C could be explored as a potential immunomodulatory agent against various solid cancers and have shown its abilities to regulate tumor microenvironment reprogramming of TAMs in a breast cancer model. Fbln7, which is a secreted glycoprotein, has been shown to be anti-angiogenic and has an immunomodulatory role regulating various functional properties of monocytes, macrophages, and neutrophils, thereby influencing inflammation. In this study, the authors have shown that in a murine breast tumor model, intravenous administration of Fbln7-C significantly reduces the size of tumors via macrophage reprogramming. Comment on: https://doi.org/10.1111/febs.15333.

A bilayered skin substitute developed using an eggshell membrane crosslinked gelatin-chitosan cryogel.

Commercially available allografts and xenografts pose problems such as high cost, risk of infection transmission and immune rejection of grafts. Thus, bioengineered skin substitutes fabricated from natural biomaterials or synthetic polymers are currently the focus of skin tissue engineering. In this study, eggshell membrane (ESM) powder was used to crosslink a gelatin-chitosan cryogel thereby replacing glutaraldehyde, a known cytotoxic chemical crosslinker. The resultant ESM-crosslinked macroporous cryogel with a pore size ranging between 10 and 350 µm has improved flexibility, biodegradability and biocompatibility compared to a glutaraldehyde-crosslinked cryogel. For healing of large and deep wounds, bilayered scaffolds which exhibit key aspects of skin physiology are being explored. Hence, we fabricated a bilayered substitute by coupling the ESM-crosslinked cryogel (dermal equivalent) to a non-porous, physically-crosslinked gelatin-chitosan film (epidermal equivalent). The epidermal layer provides the requisite barrier properties while the dermal layer facilitates cell attachment and migration for optimal wound healing. Further, chitosan confers antibacterial properties to the cryogel with almost 50% reduction in bacterial viability. Animal studies confirm that the developed bilayered skin substitute is non-allergic, aids wound healing by improving re-epithelialization within 14 days and supports the formation of skin appendages. This system presents a new and alternative treatment option for burn and chronic wounds.

Three-dimensional cryogel matrix for spheroid formation and anti-cancer drug screening.

Spheroid-based systems have been developed as alternatives to two-dimensional (2D) monolayer cultures for understanding 3D cell behavior and conducting in vitro drug screening tests. However, spheroids are easily disrupted while handling and do not mimic the presence of extracellular matrix (ECM) components. To address that, we have developed a cost-effective, polyethylene glycol diacrylate (PEGDA), and gelatin methacryloyl (GELMA) based semi-synthetic cryogel matrix system, which can be used to grow spheroids and conduct studies while providing architectural support and mimicking in vivo ECM components. These matrices are macroporous and support formation of tumor-like spheroids of breast tumor epithelial (MCF-7) cells in the absence of additional growth factors otherwise required for spheroid formation. Difference in morphology of cells as a function of matrix composition and increase in size and number of spheroids as a function of time was observed. Spheroids grown in cryogel matrices showed more drug resistance than their 2D counterparts, which can partially be explained by the epithelial to mesenchymal transition (EMT) observed in spheroids. We believe that spheroids formed through these PEGDA-GELMA cryogel matrices better represent in vivo pathological conditions and can help develop cost-effective in vitro assays for screening new pharmacological drug candidates and performing cell mechanistic studies.

Glycated collagen - a 3D matrix system to study pathological cell behavior.

A hyperglycemic condition like diabetes in patients renders them with an increased risk of developing breast cancer. Hyperglycemia and ageing increase the non-enzymatic glycosylation (glycation) of nearly all proteins in our body including collagen type I, which is an important extracellular matrix (ECM) component. This results in the formation of advanced glycated end products (AGEs), which can form covalent crosslinks in collagen fibers and change the overall architecture and stiffness of the matrix. In this study we have used MDA-MB-231 breast cancer cells to study the interaction of tumor cells with glycated collagen and have explored the role of matrix architecture and RAGE-mediated signaling in cellular behavior. We mimicked the non-enzymatic glycation of protein by treating collagen I with glucose or ribose and found that crosslinking due to AGEs induces collagen fiber bundling and an increase in pore size and stiffness of the matrix. We also observed that AGE formation triggers AGE-RAGE signaling playing a role in the morphology and migration of cells. Furthermore, our study suggests an interplay of the pore size of the collagen matrix and RAGE mediated signaling in 3D invasion of cells and our findings demonstrate that the effect of the AGE-RAGE interaction is more pronounced than that of an altered matrix architecture. This study has helped us develop a 3D system using glycated collagen to study the effects of pathological conditions such as diabetes on extracellular matrix proteins, which may have downstream effects on cell behavior and dysfunction.

Gelatin-Based Matrices as a Tunable Platform To Study in Vitro and in Vivo 3D Cell Invasion.

Hydrogels have been used as synthetic mimics of 3D extracellular matrices (ECM) and their physical properties like stiffness, degradability, and porosity have been known to influence the behavior of encapsulated cells. However, to understand the role of individual properties, the influence of biophysical cues should be decoupled from biochemical ones. In this study, we have used hydrogels as a tunable model matrix to develop a 3D cell culture platform for studying cell invasion. Inert polyethylene (glycol) diacrylate (PEGDA) and cell adhesive gelatin methacryloyl (GELMA) were blended in varying compositions, followed by UV-mediated photo polymerization to obtain hydrogels with varying stiffness, degradation, and cell adhesive properties. We developed two hydrogel matrix systems, namely, PEGDA-GELMA (containing a larger proportion of PEGDA) and GELMA-PEGDA (containing predominantly GELMA), and characterized them for differences in pore size, swelling ratio, storage modulus, degradability, and biocompatibility of the matrix. Both hydrogel systems had similar pore dimensions and swelling behavior, but PEGDA-GELMA was found to be stiffer and nondegradable, while GELMA-PEGDA was softer and degradable. Accordingly, MDA-MB-231 breast cancer cells encapsulated in these matrices showed a spheroidal morphology in PEGDA-GELMA hydrogels and were more spindle-shaped in GELMA-PEGDA hydrogels, confirming that size and extent of spreading of cells were influenced by the type of these hydrogels. The softer GELMA-PEGDA matrices readily allowed invasion of MDA-MB-231 cells in 3D and showed differences in epithelial-mesenchymal transition (EMT) gene expression of these cells. We further demonstrated the invasion and sprouting of endothelial cells using a chick aortic arch assay, exhibiting the utility of softer matrices to study 3D cell invasion for multiple applications. We also implanted these matrices in mice and showed that soft gelatin-based hydrogels allow cell infiltration in vivo. Results from our study highlight the tunability of this matrix system and the role of matrix constitution in influencing cell invasion in a 3D microenvironment.