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Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors.
Collagen density defines 3D migration of CTLs and their consequent cytotoxicity against tumor cells
(2021)
Solid tumors are often characterized by condensed extracellular matrix (ECM). The impact of dense ECM on cytotoxic T lymphocytes (CTL) function is not fully understood. Here, we report that CTL-mediated cytotoxicity is substantially impaired in dense collagen matrices. Although the intrinsic killing machinery including expression of cytotoxic proteins and degranulation was intact, CTL motility was substantially compromised in dense collagen. We found that for 3D CTL migration, persistence and velocity was regulated by collagen stiffness and the porosity, respectively. Interestingly, 3D CTL velocity is strongly correlated with their nuclear deformability/flexibility during migration, which is regulated by the microtubule network. Moreover, CTL migration was completely abolished by inhibition of actin polymerization and or myosin IIA. Remarkably, disruption of the microtubule-networks significantly improves the impaired migration, search efficiency, and cytotoxicity of CTLs in dense collagen. Our work suggests the microtubule network as a promising target to rescue impaired CTL killing capacity in solid tumor related scenarios.
Hsp47 is a chaperone protein with a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. A light-responsive Hsp47 recombinant protein, engineered to control in situ the production and assembly of cellular collagen is here demonstrated. This novel light-driven tool enables unprecedented fundamental studies of collagen biosynthesis and associated diseases.
Exogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro
(2020)
Collagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders this stability is lost, either due to mutations in collagens or in the chaperones involved in collagen assembly. This leads to chronic wounds, skin fragility, and blistering. Existing approaches to treat such conditions rely on administration of small molecules to simulate collagen production, like 4-phenylbutyrate (4-PBA) or growth factors like TGF-β. However, these molecules are not specific for collagen synthesis, and result in unsolicited side effects. Hsp47 is a collagen-specific chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen deposition. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) to skin cells, including specific collagen subtypes quantification.
Tissue regeneration and remodeling after damage requires enhanced collagen deposition at the site of damage. In collagen disorders like keratoconus and brittle bone disease this ability is lost due to collagen misfolding, poor crosslinking and deposition. For this purpose, tools that allow to control and regulate collagen biosynthesis and folding are required. Ideally, such tools should be collagen-specific and allow remote control, which available strategies fail to fulfill.In this context, a collagen-specific molecular chaperone, Hsp47, was chosen as it has multiple roles in collagen biosynthesis. Recombinant Hsp47 can be delivered in the endoplasmic reticulum of mammalian cells via KDEL receptor mediated endocytosis. Exogenous delivery of Hsp47 stimulates fibrillar collagen I, III and V in cells. A photoactivatable derivative of Hsp47 (H47Y<ONBY)was developed containing an un‐natural light‐responsive tyrosine (o‐nitro benzyl tyrosine (ONBY)),which renders Hsp47 inactive toward collagen binding. On-demand, localized and in situ activation of this tool, stimulating collagen production in disease-state cells, was tested in vitro.Also, this tool can be easily delivered precisely in cells of damage corneal tissue from keratoconus patients. Site-selective exposure after H47Y<ONBY treatment,allowing localized remodeling of the extracellular collagen matrix, was demonstrated ex vivo. This tool has potential to trigger collagen deposition in collagen deficient disorders.Tissue regeneration and remodeling after damage requires enhanced collagen deposition at the site of damage. In collagen disorders like keratoconus and brittle bone disease this ability is lost due to collagen misfolding, poor crosslinking and deposition. For this purpose, tools that allow to control and regulate collagen biosynthesis and folding are required. Ideally, such tools should be collagen-specific and allow remote control, which available strategies fail to fulfill.In this context, a collagen-specific molecular chaperone, Hsp47, was chosen as it has multiple roles in collagen biosynthesis. Recombinant Hsp47 can be delivered in the endoplasmic reticulum of mammalian cells via KDEL receptor mediated endocytosis. Exogenous delivery of Hsp47 stimulates fibrillar collagen I, III and V in cells. A photoactivatable derivative of Hsp47 (H47Y<ONBY)was developed containing an un‐natural light‐responsive tyrosine (o‐nitro benzyl tyrosine (ONBY)),which renders Hsp47 inactive toward collagen binding. On-demand, localized and in situ activation of this tool, stimulating collagen production in disease-state cells, was tested in vitro.Also, this tool can be easily delivered precisely in cells of damage corneal tissue from keratoconus patients. Site-selective exposure after H47Y<ONBY treatment,allowing localized remodeling of the extracellular collagen matrix, was demonstrated ex vivo. This tool has potential to trigger collagen deposition in collagen deficient disorders.
Abstract Collagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.
Abstract Despite the progress in surgical techniques and antibiotic prophylaxis, opportunistic wound infections with Bacillus cereus remain a public health problem. Secreted toxins are one of the main factors contributing to B. cereus pathogenicity. A promising strategy to treat such infections is to target these toxins and not the bacteria. Although the exoenzymes produced by B. cereus are thoroughly investigated, little is known about the role of B. cereus collagenases in wound infections. In this report, the collagenolytic activity of secreted collagenases (Col) is characterized in the B. cereus culture supernatant (csn) and its isolated recombinantly produced ColQ1 is characterized. The data reveals that ColQ1 causes damage on dermal collagen (COL). This results in gaps in the tissue, which might facilitate the spread of bacteria. The importance of B. cereus collagenases is also demonstrated in disease promotion using two inhibitors. Compound 2 shows high efficacy in peptidolytic, gelatinolytic, and COL degradation assays. It also preserves the fibrillar COLs in skin tissue challenged with ColQ1, as well as the viability of skin cells treated with B. cereus csn. A Galleria mellonella model highlights the significance of collagenase inhibition in vivo.
