Ca2+ sonotransfer into breast cancer cells in a suspension, 3-D spheroid and subcutaneous tumor models

Abstract

Calcium-based treatments have gained considerable attention in the field of electroporation, primarily, due to their comparable efficacy to conventional electro-chemotherapy. However, their applications in sonoporation remain under-investigated, despite its high potential for site-specific and temporally-controlled drug delivery. Current study examines the curative potential of calcium sonoporation across multiple experimental models, including: i) cell suspension, ii) 3-D spheroid culture and iii) subcutaneous murine breast cancer tumors. Murine breast cancer is an established analogue of stage IV human breast cancer. For comparison, parallel experiments, using classical anticancer drug bleomycin were performed. Ca²⁺ sonoporation efficiently enhanced 4 T1 cell death in a suspension in the absence of microbubbles, under relatively low acoustic pressure (100-200 kPa). In contrast, efficient spheroid growth reduction required microbubble-mediated inertial cavitation at higher (700 kPa) acoustic pressure. In vivo, Ca²⁺ sonoporation demonstrated similar tumor growth reduction as bleomycin sonoporation. Both treatments reduced tumor growth from the third day after the onset of treatment. Successful cancer treatment was achieved even at lower values of cavitation dose metrics. Our study presents a multi-level validation of Ca²⁺ sonoporation as an effective treatment strategy for murine breast cancer. Importantly, complete tumor eradication and prolonged animal survival up to one month were observed even at significantly reduced cavitation activity, indicating clinical safety of the treatment.

Keywords: 4T1 cells; Bleomycin; Breast cancer; Calcium; Sonoporation; Spheroids.

Graphical abstract

Fig. 1

Visual representation of US exposure setups, used for SP of cells in a suspension (A) and 3-D cell spheroids (B). Illustration of passive cavitation detection system, applied for the monitoring of MB cavitation at in vitro conditions (C). Abbreviations: HV – high voltage; RF – radio-frequency; OD – optical density.

Fig. 2

Ca²⁺ and BLM sonotransfer into 4 T1 cells in a suspension. Cell viability was evaluated after 15 min (PI assay), 48 h (MTT assay) and 6 days (CA). Cell viability for Ca²⁺ alone (0 kPa) (A1); Ca²⁺ + US (B1) and Ca²⁺ + MB + US (C1) at 50–200 kPa acoustic pressure; cell viability data for Ca²⁺ + US and Ca²⁺ + MB + US, grouped according to acoustic pressure (D1). Cell viability for BLM alone (0 kPa) (A2); BLM + US (B2) and BLM + MB + US (C2) at 50–200 kPa acoustic pressure; cell viability data for BLM + US and BLM + MB + US, grouped according to acoustic pressure (D2). “NS” – not significant; * – p < 0.05; ** – p < 0.01; *** – p < 0.001.

Fig. 3

4 T1 cell viability for US, Ca²⁺ + US, MB + US and Ca²⁺ + MB + US at 50–200 kPa acoustic pressure. Cell viability was evaluated after 15 min (PI assay), 48 h (MTT assay) and 6 days (CA). “NS” – not significant; * – p < 0.05; ** – p < 0.01; *** – p < 0.001.

Fig. 4

Ca²⁺ sonotransfer to 3-D culture of 4 T1 cells. 4 T1 cell spheroids in agarose gel (A). The results of spheroid growth for different experimental groups at 400 (B) and 700 (C) kPa acoustic pressure. Microscopical images of spheroids: 1- control; 2- Ca²⁺; 3- Ca²⁺ + US (700 kPa); 4- MB + US (700 kPa); 5- Ca²⁺ + MB + US (700 kPa). “NS” – non-significant; * – p < 0.05; ** – p < 0.01; *** – p < 0.001.

Fig. 5

The dynamics of US side-scattered signals and MB concentration. Representative side-scattered US pulses (A). Processed, filtered (4th order Butterworth filter) and averaged (n = 4 different experiments) time–frequency-amplitude colormaps: i) for MB-containing (+MB) groups at 100–700 kPa; ii) MB-free (−MB) group for 200 kPa (B). RMS curves, quantified in 1.5–1.75 MHz frequency range, for low 50 and 100 kPa acoustic pressures (C). RMS dynamics, evaluated across different frequency ranges, for 200 kPa acoustic pressure (D). RMS curves, quantified in 1.5–1.75 MHz frequency range, for 300, 400 and 700 kPa acoustic pressures (E). Normalised RMS curves for 300 kPa, quantified in frequency range of ± 0.1 MHz around: the 2nd harmonic (2f), 1st ultraharmonic (3/2f) and 1st subharmonic (1/2f); f = 1 MHz (F). Micrographs, illustrating MB concentration decrease, at 200 kPa acoustic pressure (G) and corresponding OD values, representing MB concentration decrease (H). Temporal MB concentration decrease for 50–700 kPa acoustic pressures (I).

Fig. 6

Ca²⁺ and BLM sonotransfer into subcutaneous tumors of 4 T1 breast cancer cells. Mouse image, representing the site of healed tumor (yellow arrow) after Ca²⁺ SP (A). The dynamics of tumor volume during post-treatment period (B). Quantification of tumor volume reduction rate using exponential curve fitting (C); tumor reduction rate for Ca²⁺ SP and BLM SP (C inset). Mouse survivability, illustrated by Kaplan-Meyer curves for each experimental group (D).

Fig. 7

Schematic representation of US-side scattered signal registration (A). Frequency spectra for MB-containing (MB + US) and MB-free (US alone) groups (B), shown for the frame #634 (41.3 s); the inset highlights frequency spectra within 1.5–1.75 MHz frequency range. RMS curves for + MB and − MB groups, quantified in 1.5–1.75 MHz frequency range (C); frame #634 is indicated by blue arrows. Temporal increase in FFT amplitude in 1.5–1.75 MHz frequency range (D). Temporal decrease in FFT amplitude for the 2nd (2f), 3rd (3f) and 4th (4f) harmonics (f = 0.95 MHz) (E). RMS curves, evaluated in 1.5–1.75 MHz frequency range and its corresponding repetitive ranges at higher frequencies (F). RMS curves, quantified within ± 0.1 MHz frequency band around the 2nd (2f), 3rd (3f) and 4th (4f) harmonics (G). RMS curves, calculated within ± 0.1 MHz frequency band around the 1st suharmonic (1/2f) and 1st ultraharmonic (3/2f) (H). Examples of the variability in RMS curves (1.5–1.75 MHz), obtained from different tumor samples (I). The correlation between CD and tumor decrease rate, pooled for Ca²⁺ SP and BLM SP (J).

Fig. 8

US diagnostic imaging of subcutaneous tumors. A gradual reduction in the intensity of US back-scattered signal in MB bolus (orange arrow in upper panel) or ROI (red circle in lower panel) areas, followed by simultaneous disappearing of MB shadow (yellow arrow) (A). Normalised B-scan intensity curves with respective sigmoidal approximations, obtained from different tumors (B). The correlation between MB sonodestruction rate and tumor decrease rate (C).

Fig. 9

The correlation between MB sonodestruction rate and CD.