Mechanical manipulation of cancer cell tumorigenicity via heat shock protein signaling

Abstract

Biophysical cues of rigid tumor matrix play a critical role in cancer cell malignancy. We report that stiffly confined cancer cells exhibit robust growth of spheroids in the stiff hydrogel that exerts substantial confining stress on the cells. The stressed condition activated Hsp (heat shock protein)–signal transducer and activator of transcription 3 signaling via the transient receptor potential vanilloid 4–phosphatidylinositol 3-kinase/Akt axis, thereby up-regulating the expression of the stemness-related markers in cancer cells, whereas these signaling activities were suppressed in cancer cells cultured in softer hydrogels or stiff hydrogels with stress relief or Hsp70 knockdown/inhibition. This mechanopriming based on three-dimensional culture enhanced cancer cell tumorigenicity and metastasis in animal models upon transplantation, and pharmaceutically inhibiting Hsp70 improved the anticancer efficacy of chemotherapy. Mechanistically, our study reveals the crucial role of Hsp70 in regulating cancer cell malignancy under mechanically stressed conditions and its impacts on cancer prognosis–related molecular pathways for cancer treatments.

Previously described methods were used to fabricate MeHA (18) with ~15, ~50, and ~115% methacrylation from sodium hyaluronate (weight-average molecular weight of 40 to 70 kDa; Lifecore). The chemical structures of the modified HA polymers were evaluated by 1H nuclear magnetic resonance (Bruker) spectra. A total of 7.2 mg GCGYGRGDSPG or nonbioactive GCGYGRADSPG (GenScript) was coupled with 100 mg of MeHA overnight reaction in pH 8.0 at 25°C for presenting integrin binding moiety with subsequent lyophilization. Primary CRCs or cancer cell lines including HeLa [the American Type Culture Collection (ATCC), cat. no. CCL-2, RRID:CVCL_0030], MDA-MB-231 (ATCC, cat. no. HTB-26, RRID:CVCL_0062), MCF-7 (ATCC, cat. no. HTB-22, RRID:CVCL_0031), Hep3B (ATCC, cat. no. HB-8064, RRID:CVCL_0326), CT26 cells (ATCC, cat. no. CRL-2638, RRID:CVCL_7256), or passage 3 hMSC (male; Lonza) were encapsulated into MeHA hydrogel (106 cells ml−1) using photoinitiated free radical polymerization (EXFO/OmniCure S1000 lamp with a 320- to 390-nm filter, exposure of 10 mW cm−2 for 5 min) with 0.05 weight % Irgacure 2959 (I2959, Ciba), a photoinitiator chosen for its aqueous solubility and good cytocompatibility (19). The initial cell seeding density was kept the same among all groups of hydrogels. The elastic modulus of the hydrogels was measured by parallel-plate compression testing at 10% ramped strain min−1 as previously reported (18). All gels encapsulating cells were transferred to growth media supplemented with Dulbecco's modified Eagle medium (Thermo Fisher Scientific), 10% fetal bovine serum (Invitrogen), 1 mM l-glutamine (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 0.1 mM penicillin/streptomycin (Invitrogen) at 37°C and 5% CO2. Media changes were performed every 3 days. For the compressive stress study, the cell-containing gel was in a 35-mm2 culture dish (Corning) in Mach-1 Micromechanical System (Biomomentum Inc., model no. MA005) at 37°C and 5% CO2. Uniaxial unconfined strain (ε) from 0.05 to 0.35 was imposed onto the gel with a flat indenter of 25.4 mm in diameter. The duration of compression for all cellular uptake experiments was fixed over 72 hours and fixed for further analysis.