Tumor Stiffening, a Key Determinant of Tumor Progression, is Reversed by Nanomaterial-Induced Photothermal Therapy

Abstract

Tumor stiffening, stemming from aberrant production and organization of extracellular matrix (ECM), has been considered a predictive marker of tumor malignancy, non-invasively assessed by ultrasound shear wave elastography (SWE). Being more than a passive marker, tumor stiffening restricts the delivery of diagnostic and therapeutic agents to the tumor and per se could modulate cellular mechano-signaling, tissue inflammation and tumor progression. Current strategies to modify the tumor extracellular matrix are based on ECM-targeting chemical agents but also showed deleterious systemic effects. On-demand excitable nanomaterials have shown their ability to perturb the tumor microenvironment in a spatiotemporal-controlled manner and synergistically with chemotherapy. Here, we investigated the evolution of tumor stiffness as well as tumor integrity and progression, under the effect of mild hyperthermia and thermal ablation generated by light-exposed multi-walled carbon nanotubes (MWCNTs) in an epidermoid carcinoma mouse xenograft. SWE was used for real-time mapping of the tumor stiffness, both during the two near infrared irradiation sessions and over the days after the treatment. We observed a transient and reversible stiffening of the tumor tissue during laser irradiation, which was lowered at the second session of mild hyperthermia or photoablation. In contrast, over the days following photothermal treatment, the treated tumors exhibited a significant softening together with volume reduction, whereas non-treated growing tumors showed an increase of tumor rigidity. The organization of the collagen matrix and the distribution of CNTs revealed a spatio-temporal correlation between the presence of nanoheaters and the damages on collagen and cells. This study highlights nanohyperthermia as a promising adjuvant strategy to reverse tumor stiffening and normalize the mechanical tumor environment.

Keywords: cancer; carbon nanotubes; elastography; photothermal therapy; tumor microenvironment..

Figure 1

Photothermal treatment mediated by CNTs. (a) Picture of a near-infrared irradiation (808 nm, 1 W/cm²) set-up in vivo with laser beam focusing on the tumor area. (b) Thermographic infrared pictures of a mouse under laser exposure during thermal ablation at four time points; right scale representing the color code for surface temperature. (c) Surface temperature plot as a function of the time for thermal ablation (T.A., in black - 3 min) and mild hyperthermia (M.H.T., in red - 15 min) and the related non-specific tissue heating for non-injected tumors for 3 min.

Figure 2

Real-time SWE mapping and quantification of tumor stiffness during thermal therapy. (a) Picture of the experimental set-up with near-infrared irradiation (IR) (808 nm, 1-2 W/cm²) with simultaneous SWE in vivo. (b) Normalized mean tumor stiffness as a function of the time featuring laser exposure windows for mild hyperthermia group (n=6). (c) Normalized mean tumor stiffness plot as a function of the time featuring laser exposure windows for thermal ablation group (n=12). (d) Histogram illustrating the shift of the stiffness distribution for the tumor represented in (e), at three time points before, during and after the first irradiation of thermal ablation treatment (IR1). (e) SWE color stiffness mapping at three time points before, during and after the first irradiation of thermal ablation. (f) Histogram of the stiffness distribution of the same tumor before the first irradiation of the thermal ablation treatment (IR1) and before the second irradiation (IR2) showing the reversibility of laser-induced stiffening during IR1. (g) Histogram of the stiffness distribution for the control non-injected tumor represented in (h) at three time points before, during and after IR1 at the laser set-up corresponding to thermal ablation. (h) SWE color stiffness mapping at three time points for a control tumor before, during and after IR1.

Figure 3

Photothermal therapy effect on tumor growth. (a) Tumor volume plot as a function of the time for control (n=14), mild hyperthermia (n=6) and thermal ablation (n=14) groups. Control group comprises non-CNT injected non-irradiated tumors (n=6) and non-CNT injected tumors irradiated with laser fluence used for mild hyperthermia (n=4) and thermal ablation (n=4). (b) Representative pictures of tumor growth evolution for control, mild hyperthermia and thermal ablation groups (The panel with the cross represents a sacrificed mouse whose tumor mass achieved ethical limit).

Figure 4

Evolution of the tumor stiffness following treatment. SWE color maps of stiffness (range 0-40 kPa) with corresponding B-mode view in the transverse plane of control (a) and treated (b) tumor over 9 days. Color scale is the same for all measurements and scale bar corresponds to 5 mm. The white arrow (D9, treated) indicates the location of the residual tumor. (c) Quantitative analysis of the normalized mean tumor stiffness for 9 days for control tumors (n=8) and CNT-injected tumors treated either by thermal ablation (n=12) or mild hyperthermia (n=6). The mean stiffness was normalized to the baseline value at day 0 before the first irradiation for each tumor (***p≤0.0004). (d) Quantitative analysis of the percentage of intratumoral area above the threshold of 40 kPa for three time points after treatment. Tumors treated by thermal ablation or mild hyperthermia were classified into treatment responsive (n=9) and non-responsive (n=7), as a function of the tumor volume decrease or increase following thermal therapy, respectively (**p=0.05 and ***p=0.002).

Figure 5

Histological analysis of CNT-injected non-irradiated control, thermal ablation and mild hyperthermia groups. Control group of CNT-injected non-irradiated tumor (a-b). High magnification view of tumor cell mass featured carbon nanotubes among tumor cells (a). Carbon nanotubes were visualized as dark spots near collagen fibers (left white arrows) and in the tumor mass featuring inflammatory cells (center and right white arrows) (b). Tumor 1 h following thermal ablation (c-d) featuring an extended necrotic zone (red arrow) including hemorrhagic necrosis (blue arrow) (c). Necrotic tissue at higher magnification (d). Tumor 2 days following thermal ablation featuring an extended necrotic zone (red arrow) including hemorrhagic necrosis (blue arrow) with a zone featuring a large amount of carbon nanotubes (white star) (e). Tumor 1 h following mild hyperthermia (f-g) featuring an extended necrotic zone (red arrow) including hemorrhagic necrosis (blue arrow) (f). Hemorrhagic necrotic tissue (blue arrow) at higher magnification (g).

Figure 6

Collagen destructuration analysis by SHG in tumor slices in response to CNT-mediated PTT (a) Images of only collagen fibers were obtained by substracting the 850 nm excitation images to 810 nm ones. Images of only CNTs were acquired at 850 nm excitation wavelength. Images of both collagen and CNTs were obtained at 810 nm excitation wavelength. Intact collagen fibers were observed for the non CNT-injected non-irradiated control, the non CNT-injected irradiated control as well as for the CNT-injected non-irradiated control (first, second and third lines). For the latter, CNTs were visualized as bright spots (third line). For the CNT-injected and irradiated group (thermal ablation condition, 1 day time point in the fourth line), few intact collagen fibers were observed in zones devoid of CNTs while destructurated collagen fibers were visualized nearby CNTs (circled region). (b) Pearson's coefficient demonstrating the correlation between CNTs and collagen destructuration (n=12 slices from 3 different tumors for each condition, TA (MH) 1D: thermal ablation (respectively, mild hyperthermia) at day 1 post-irradiation, TA (MH) 30 min: thermal ablation (respectively, mild hyperthermia) 30 min post-irradiation).

Figure 7

Cell damage evaluation by TPEF and SHG microscopy in response to CNT-mediated PTT in tumor slices. Each image is a reconstitution of 6×5 stitched pictures viewed in volume with the software Imaris BitPlan (8 steps of 15 µm). Circled regions indicate the presence of CNTs. Without laser irradiation, the integrity of the tissue was observed both in the presence or absence of CNTs (a) for CNT-injected non-irradiated control group. Under laser exposure, tissue damage correlated with the presence of CNTs while the tissue preserved its integrity in the regions devoid of CNTs for CNT-injected and irradiated group (b). Distribution of cell autofluorescence levels in TPEF images of tumor slices from non CNT-injected non-irradiated and CNT-injected non-irradiated conditions (c) as well as non CNT-injected irradiated and CNT-injected irradiated (thermal ablation at day 1) conditions (d). A left-shift of cell autofluorescence levels was observed following PTT indicating cell damage in comparison to all three control conditions. Average intensity of cell autofluorescence in TPEF images of tumor slices from non CNT-injected non-irradiated; CNT-injected non-irradiated conditions; non CNT-injected irradiated and CNT-injected irradiated (thermal ablation at day 1) conditions (e). A statistically significant nearly 2-fold decrease in cell signal was induced by photothermal treatment indicating cell damage (n=12 slices from 3 different tumors for each condition).