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Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes.

ABSTRACT: Lymphocyte motility is governed by a complex array of mechanisms, and highly dependent on external microenvironmental cues. Tertiary lymphoid sites in particular have unique physical structure such as collagen fiber alignment, due to matrix deposition and remodeling. Three dimensional studies of human lymphocytes in such environments are lacking. We hypothesized that aligned collagenous environment modulates CD8+ T cells motility. We encapsulated activated CD8+ T cells in collagen hydrogels of distinct fiber alignment, a characteristic of tumor microenvironments. We found that human CD8+ T cells move faster and more persistently in aligned collagen fibers compared with nonaligned collagen fibers. Moreover, CD8+ T cells move along the axis of collagen alignment. We showed that myosin light chain kinase (MLCK) inhibition could nullify the effect of aligned collagen on CD8+ T cell motility patterns by decreasing T cell turning in unaligned collagen fiber gels. Finally, as an example of a tertiary lymphoid site, we found that xenograft prostate tumors exhibit highly aligned collagen fibers. We observed CD8+ T cells alongside aligned collagen fibers, and found that they are mostly concentrated in the periphery of tumors. Overall, using an in vitro controlled hydrogel system, we show that collagen fiber organization modulates CD8+ T cells movement via MLCK activation thus providing basis for future studies into relevant therapeutics.

SUBMITTER: Pruitt HC

PROVIDER: S-EPMC6697628 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes.

Pruitt Hawley C HC Lewis Daniel D Ciccaglione Mark M Connor Sydney S Smith Quinton Q Hickey John W JW Schneck Jonathan P JP Gerecht Sharon S

Matrix biology : journal of the International Society for Matrix Biology 20190216

Lymphocyte motility is governed by a complex array of mechanisms, and highly dependent on external microenvironmental cues. Tertiary lymphoid sites in particular have unique physical structure such as collagen fiber alignment, due to matrix deposition and remodeling. Three dimensional studies of human lymphocytes in such environments are lacking. We hypothesized that aligned collagenous environment modulates CD8+ T cells motility. We encapsulated activated CD8+ T cells in collagen hydrogels of d ...[more]

PMID: 30776427

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Project description:In the tumor microenvironment (TME), collagen fibers facilitate tumor cell migration through the extracellular matrix. Previous studies have focused on studying the responses of cells on uniformly aligned or randomly aligned collagen fibers. However, the in vivo environment also features spatial gradients in alignment, which arise from the local reorganization of the matrix architecture due to cell-induced traction forces. Although there has been extensive research on how cells respond to graded biophysical cues, such as stiffness, porosity, and ligand density, the cellular responses to physiological fiber alignment gradients have been largely unexplored. This is due, in part, to a lack of robust experimental techniques to create controlled alignment gradients in natural materials. In this study, we image tumor biopsy samples and characterize the alignment gradients present in the TME. To replicate physiological gradients, we introduce a first-of-its-kind biofabrication technique that utilizes a microfluidic channel with constricting and expanding geometry to engineer 3D collagen hydrogels with tunable fiber alignment gradients that range from sub-millimeter to millimeter length scales. Our modular approach allows easy access to the microengineered gradient gels, and we demonstrate that HUVECs migrate in response to the fiber architecture. We provide preliminary evidence suggesting that MDA-MB-231 cell aggregates, patterned onto a specific location on the alignment gradient, exhibit preferential migration towards increasing alignment. This finding suggests that alignment gradients could serve as an additional taxis cue in the ECM. Importantly, our study represents the first successful engineering of continuous gradients of fiber alignment in soft, natural materials. We anticipate that our user-friendly platform, which needs no specialized equipment, will offer new experimental capabilities to study the impact of fiber-based contact guidance on directed cell migration.

Project description:BackgroundTriple negative breast cancer (TNBC) is characterized by poor prognosis and a lack of effective therapeutic agents owing to the absence of biomarkers. A high abundance of tumor-infiltrating regulatory T cells (Tregs) was associated with worse prognosis in malignant disease. Exploring the association between Treg cell infiltration and TNBC will provide new insights for understanding TNBC immunosuppression and may pave the way for developing novel immune-based treatments.Materials and methodsPatients from TCGA were divided into Treg-high (Treg-H) and Treg-low (Treg-L) groups based on the abundance of Tregs according to CIBERSORT analysis. The association between expression level of Tregs and the clinical characteristics as well as prognosis of breast cancer were evaluated. Next, a Treg-related prognostic model was established after survival-dependent univariate Cox and LASSO regression analysis, companied with an external GEO cohort validation. Then, GO, KEGG and GSEA analyses were performed between the Treg-H and Treg-L groups. Masson and Sirius red/Fast Green staining were applied for ECM characterization. Accordingly, Jurkat T cells were encapsulated in 3D collagen to mimic the ECM microenvironment, and the expression levels of CD4, FOXP3 and CD25 were quantified according to immunofluorescence staining.ResultsThe expression level of Tregs is significantly associated with the clinical characteristics of breast cancer patients, and a high level of Treg cell expression indicates a poor prognosis in TNBC. To further evaluate this, a Treg-related prognostic model was established that accurately predicted outcomes in both TCGA training and GEO validation cohorts of TNBC patients. Subsequently, ECM-associated signaling pathways were identified between the Treg-H and Treg-L groups, indicating the role of ECM in Treg infiltration. Since we found increasing collagen concentrations in TNBC patients with distant migration, we encapsulated Jurkat T cells within a 3D matrix with different collagen concentrations and observed that increasing collagen concentrations promoted the expression of Treg biomarkers, supporting the regulatory role of ECM in Treg infiltration.ConclusionOur results support the association between Treg expression and breast cancer progression as well as prognosis in the TNBC subtype. Moreover, increasing collagen density may promote Treg infiltration, and thus induce an immunosuppressed TME.

Project description:Vitamin D3 (vitD3) has been implicated in various cellular functions affecting multiple tissue types. Epidemiological and laboratory studies suggest that vitD3 may be effective as a preventive or therapeutic option for breast cancer. However, randomized clinical trials have yet to confirm these suggestions. Breast neoplasias can arise from developmental alterations; based on this evidence, we seek to understand vitD3's role in normal breast development, particularly its role in epithelial morphogenetic processes such as ductal elongation, branching, and alveolar formation. These processes require extensive changes in the extracellular microenvironment, such as collagen fiber organization, and are largely influenced by hormones. Here, we build upon our past work to shed light on calcitriol's effects on collagen fiber organization by breast epithelial cells, and how such effects are modulated by extracellular matrix composition. We embedded MCF10A normal human breast epithelial cells in two different matrices-collagen type I and collagen type I + 10% Matrigel; treatment with calcitriol resulted in flatter epithelial structures. Next, using two-photon microscopy, we examined changes in collagen fiber organization and corresponding changes in epithelial structures. Applying a novel three-dimensional (3D) image analysis method, we show that increasing doses of calcitriol result in denser collagen fiber bundles in the localized area surrounding the epithelial structures, and that these bundles are aligned in a more parallel direction to epithelial structures when exposed to the highest vitD3 dose. Changed patterns in fiber organization may explain the flattening of epithelial structures; in turn, changes in biophysical forces in the matrix abutting these structures may be responsible for changes in the referred patterns. Addition of 10% Matrigel dampened the effects of calcitriol on both epithelial morphogenesis and fiber organization. Overall, we report novel functions of calcitriol in the breast epithelium and add to the growing body of evidence documenting how hormones affect biophysical processes. Impact statement In this study, we report novel functions of calcitriol in the breast epithelium and use a novel quantitative metric to parse the effects of calcitriol on collagen fiber organization that cannot be detected through conventional histological procedures. Despite the large body of literature on vitamin D3 (vitD3) and calcitriol's effects on cellular functions across tissue types, little is known about how they affect collagen fiber organization, an early critical step for breast epithelial development. This work provides further evidence that hormones affect morphogenesis by means of biophysical forces, with implications for a comprehensive view on vitD3's effects in breast development and neoplasia.

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Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes.

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Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes.

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