Microenvironment Rheology Modulates the Effect of the Anticancer Peptide CIGB300 on 3D Head and Neck Tumoroids

TL;DR

This study shows how the stiffness of a 3D tumor environment affects the performance of an anticancer peptide called CIGB300.

Contribution

The first investigation of CIGB300's diffusion in tunable hydrogels using fluorescence spectroscopy for drug screening.

Findings

The antitumor activity of CIGB300 varies with the stiffness of the 3D tumoroid microenvironment.

Fluorescence spectroscopy effectively tracks CIGB300's diffusion in hydrogels over time.

CIGB300's structural and stability properties were characterized using spectroscopic methods.

Abstract

3D cell systems for in vitro experimental studies are able to mimic the in vivo efficacy of drugs before they are tested on animals. However, many studies are still needed in order to mimic the physiological environment with 3D cell-growth systems. The mechano-physical properties of the microenvironment are relevant for the invasiveness of cancer cells and for their drug resistance. In this study, 3D tumoroids of human oral squamous cell carcinoma (OSCC) CAL27 cells of different stiffnesses were produced using a tunable PEG–silk fibroin hydrogel (PSF), and the antitumor activity of the peptide CIGB300, an anticancer therapeutic peptide, with respect to these 3D tumoroid models was assessed. Furthermore, spectroscopic studies on the CIGB300 peptide are reported regarding its structure, stability, aggregation and diffusion properties. For the first time, the diffusion of the peptide CIGB300 in tunable silk fibroin hydrogels of different stiffnesses is investigated over time via fluorescence spectroscopy as a potential tool in drug-screening using hydrogel-based 3D tumoroids.

Figures

Spectroscopic characterization of the peptide CIGB300. (A) Amino acid sequence of the peptide CIGB300. (a) UV-vis absorption spectra of CIGB300 recorded at different concentrations, ranging from 48 µM to 326 µM (λ = 240–340 nm), and (b) linear regression of absorbance at 280 nm at different concentrations of CIGB300. (B) Circular dichroic spectrum of CIGB300 obtained at 65.5 µM. (C) Fluorescence spectra (a) obtained at different concentrations of CIGB300 (48, 54, 65, 81, 108, 136, 145 and 163 µM) with excitation at λ_ex_ = 280 nm and emission at λ_em_ = 300–440 nm, and (b) linear regression of

Full text

1. Introduction

3D cell culture systems are suitable tools for significantly enhancing our insight into the effects of the microenvironment on cancer cells’ invasiveness and responses to mechano-physical cues and pharmacological treatments [1,2,3,4,5]. Recently, a valid alternative to simple spheroids obtainable through commercial kits, in which the cell density and rheology of the spheroid are not tunable, was presented: the use of cellular microsphere systems produced by photopolymerizable semi-synthetic hydrogels [6,7,8,9]. This strategy allows one to vary the chemical and mechano-physical properties of the system and thus assess different conditions and effects on proliferation, cell invasiveness, and the expression of genes of metabolic pathways relevant to tumorigenicity and drug resistance [10,11,12,13]. In this study, the effects of stiffness on the drug response of cancer cells were investigated on 3D PEG–silk-fibroin (PSF) hydrogel-based tumoroids using an antitumor peptide named CIGB300. This is a cyclic peptide (GRKKRRQRRRPPQ-β-alaCWMSPRHLGTC—see Figure 1A) characterized by a disulfide bond between the two cysteine residues and the presence of a cell-penetrating peptide sequence derived from the HIV-Tat protein at the N-terminus. This compound was obtained by the Center of Genetic Engineering and Biotechnology of Havana (Cuba) via screening a random cyclic peptide phage library against the HPV-16 E7 oncoprotein site targeted by CK2 for phosphorylation [14,15,16]. The anticancer activity of CIGB300 is linked to the ability of this peptide to inhibit casein kinase 2 (CK2), and it has been investigated ever since the peptide was first developed [15,16]. Several studies have demonstrated its ability to modulate critical hallmarks of tumor biology [17,18,19,20,21]. In the lung cancer line NCI-H82, CIGB300 was shown to interact with B23/nucleophosmin 1 (B23/NPM1) in the nucleolus, thereby disrupting its CK2-mediated phosphorylation and triggering rapid apoptotic cell death, a process apparently preceded by nucleolar disassembly [22,23]. Consistently, CIGB300 impaired the CK2-dependent phosphorylation of NPM1, leading to cell cycle arrest and apoptosis. Moreover, CIGB300 induced rapid, dose-dependent apoptosis in cancer cells and displayed broad antiproliferative activity within the micromolar range (20–300 μM) across lung, cervical, prostate, and colon-cancer-derived cell lines [15,16,24,25]. Moreover, intravenous and intraperitoneal administration of CIGB300 significantly reduced primary tumor growth in heterotopic models of cervical and lung cancer [24]. Consistent with these findings, CIGB300 exhibited pronounced anti-angiogenic and pro-apoptotic effects both in vitro and in vivo [26]. Importantly, these studies have provided the basis for the initiation of clinical trials on patients with cervical cancer [15,16,24,27]. Following the completion of multiple phase I clinical trials, which established the safety and tolerability of intra-tumoral administration, a phase II clinical trial is currently underway to evaluate the efficacy of CIGB300 in patients with stage IB2/II cervical cancer [28]. CIGB300 markedly reduces the adhesion, migration, and invasion capacities of human H125 and murine 3LL lung cancer cells. In vivo, studies employing the syngeneic 3LL lung cancer model in C57BL/6 mice demonstrated robust anticancer activity, including inhibition of lung colonization, metastasis, and tumor-induced neovascularization [24]. Further analyses revealed that CIGB300 impaired migration, invasion, and tumor-cell-secreted protease activity. Mechanistically, CK2’s inhibition by CIGB300 was associated with reduced motility and invasiveness of lung cancer cells, concomitant with suppression of the proteolytic activity of tumor-cell-secreted urokinase plasminogen activator (uPA) and matrix metalloproteinase2 (MMP2) [24,29]. In this study, the anti-cancer effects of CIGB300 on cell proliferation and viability of the less invasive oral squamous cell carcinoma (OSCC) cell line CAL27 were investigated in 2D and 3D (CALPSF) cell culture systems. The effects of different stiffnesses of the 3D-CALPSF system on B23/NPM1 expression and drug resistance were assessed, along with the effects of CIGB300 treatment on cancer cell viability. Moreover, the aggregation and diffusion properties of CIGB300 in the silk fibroin (SF) hydrogels were investigated using molecular spectroscopy. The diffusion of the peptide CIGB300 from a solution into the hydrogel was evaluated using fluorescence spectroscopy, which has never been done before, in order to estimate how changes in the stiffness of the hydrogel-based tumoroids affect both diffusion and cancer cells of the circular-antitumor peptide CIGB300.

2. Results and Discussion

2.1. Molecular Characterization of CIGB300’s Aggregation via UV-Vis Absorption, and Fluorescence Spectroscopy and Circular Dichroism

The UV-vis spectra of CIGB300 recorded at different concentrations, ranging from 48 µM to 326 µM, are shown in Figure 1A. The absorbance at 280 nm increased proportionally to the peptide concentration, as shown in Figure 1A(b) via linear regression. An ε_280_ molar extinction coefficient of 4468 (S.D. ± 284) M^−1^ cm^−1^ was obtained. In a water solution, the peptide showed a circular dichroic spectrum characteristic of cyclic peptides [30,31,32], with a minimum molar ellipticity at 200 nm, which is characteristic of a deconvolution of a beta strand with a turn structure (Figure 1B). Deconvolution of the CD curve using BestSel code [33] provided values of 30% for antiparallel β-sheets, 17% for β-turns, and 53% for random conformations.

The fluorescence emission spectrum of CIGB300, obtained by excitation at λ_ex_ = 280 nm, showed a maximum intensity at λ_em_ = 350 nm, characteristic of the tryptophan residue in the sequence. Also, in this case, fluorescence emission intensity increased proportionally to the peptide concentration, suggesting that peptide aggregation was negligible in the range of concentrations used. Figure 1. Spectroscopic characterization of the peptide CIGB300. (A) Amino acid sequence of the peptide CIGB300. (a) UV-vis absorption spectra of CIGB300 recorded at different concentrations, ranging from 48 µM to 326 µM (λ = 240–340 nm), and (b) linear regression of absorbance at 280 nm at different concentrations of CIGB300. (B) Circular dichroic spectrum of CIGB300 obtained at 65.5 µM. (C) Fluorescence spectra (a) obtained at different concentrations of CIGB300 (48, 54, 65, 81, 108, 136, 145 and 163 µM) with excitation at λ_ex_ = 280 nm and emission at λ_em_ = 300–440 nm, and (b) linear regression of the fluorescence intensity at 350 nm at different concentrations of CIGB300. Each spectrum was the mean of three acquisitions, and three experimental replicates were employed.

2.2. CIGB300 Affects the Cell Viability and Migration of the CAL 27 Cancer Cell Line in 2D Cultures

Firstly, the effects of the peptide CIGB300 on the cell viability of human head and neck squamous carcinoma adherent cells of the CAL 27 cancer cell line were assessed using 2D cell cultures. Figure 2A shows the cell viability of CAL 27 cells after 24 h of treatment with 0, 80, 160, 240 and 320 µM of CIGB300. A decrease in the cell viability of the CAL 27 cancer cells in a concentration-dependent manner was observed, which was significant at CIGB300 concentrations higher than 80 µM in the cell culture medium, and the estimated IC_50_ was 207.5 µM. Optical micrographs of the cells after 24 h of cell growth in the absence and presence of 160 µM of CIGB300 are shown in Figure 2B. Moreover, the peptide was also able to reduce cell migration/invasiveness in a concentration-dependent manner, as shown in Figure 2C. A significant reduction in migration was also observable after treatment with 80 µM of CIGB300 via a scratch assay; this reduction was not due to an inhibition of cell viability.

2.3. CAL 27 Tumoroid Production

PEG–silk fibroin hydrogel (PSF) was obtained using a method described in previous studies [4,34]. The viscoelastic properties of the PSFs were previously assessed using oscillatory shear rheometric analysis [4]. PSFs whose content of PEGDa (10 kDa) was 4.5% (named PSFHy_soft_) and 9% (named PSFHy_stiff_) (w/v), without embedded cells, were characterized via rheological nanoindentation methodology, showing shear storage moduli (E_eff_, effective Young’s moduli) of 3.37 (±0.03) kPa and 7.81 (±0.01) kPa, respectively (Figure 3B). The choice of these two stiffness conditions was based on previous results we obtained on breast cancer PSFHy-based tumoroids, where cell viability was not affected [4]. The higher stiffness was similar to that measured for invasive carcinoma tissue, whose stiffness typically ranges between 6 and 10 kPa [35]. Spheres of CAL 27 cancer cells in PSF (CAL27PSF) were produced using photopolymerization with UVA with drops of 3 or 5 µL of PSF precursor solution on the superhydrophobic nano-structured polydimethylsiloxane (PDMS) surface (Figure 3A).

The micrographs of the CAL 27 spheroids (CAL27PSF) are shown in Figure 3C. After 1 week of cell growth, the CAL27PSFs were stained with Hoechst 33342 under living conditions, and the fluorescence of the nuclei was observed after fixing via confocal fluorescence microscopy (see Figure 3D). Good viability was observed using a WST-1 metabolic assay after 24 h of cell culture of CAL27PSF_stiff_ (Figure 3E) and after three days of cell culture of CAL27PSF_soft_ and CAL27PSF_stiff_ via a LIVE/DEAD fluorescence assay (Figure 4A). Fluorescence micrographs of the CAL27PSF in soft and stiff systems obtained using Hoechst staining under living conditions after 4 days of culture are shown in Figure 4B. No significant differences between the two systems were observed.

However, after 3 weeks of cell culture, in CAL27PSFsoft, we observed the presence of cell clusters that were not present in CAL27PSFstiff (Figure 4C). This finding indicates that the greater stiffness of this microenvironment affects the ability of the cells to form carcinogenic cell clusters. Cyclin D1 expression was evaluated in CAL27PSFstiff produced using concentrations of 9% w/v PEGDa (Figure 4D). No significant changes in cyclin D1 expression were observed over time, indicating good invasion and proliferation of the cancer cells in this system. Moreover, the results regarding the acetylation of the histone H3 in 3D at high stiffness were also similar to what was observed for the cells grown on TCP and over time. This result is in agreement with the existing link between cyclin D1 and histone acetylation [36].

2.4. Effects of CIGB300 on the Cell Viability of CAL27PSF Tumoroids in Relation to Stiffness

The cell viability of the CAL27PSF_soft_ and CAL27PSFstiff samples treated with different concentrations of CIGB300 was assessed using a LIVE/DEAD (L/D) assay (see Figure 5B).

After 3 days of treatment, the cell viability L/D assay showed an increase in cell death depending on the CIGB300 concentration in the cell medium in both the CAL27PSF_soft_ and CAL27PSF_stiff_ samples. A major effect was observed in the CAL27PSF_stiff_ tumoroids. The cell viability of the tumoroids in the presence of CIGB300 in the cell culture medium was also evaluated using a metabolic assay (Figure 5C). We observed a 24.2% decrease (S.D. ± 6.59) in the cell viability of the CAL27PSF_stiff_ tumoroids after 72 h of treatment with 80 µM of CIGB300.

2.5. Diffusion Studies on CIGB300 in SF Hydrogels Conducted Using Fluorescence Spectroscopy

Tunable rheological cellular models allow one to mimic the complexity of natural architectures and physiological drug administration. The diffusion of molecules in a hydrogel-based tumoroid has a crucial role in drug administration. In general, the majority of the drug permeation mechanism is mediated by diffusion kinetics, and it is mainly considered to be Fickian [37]. In this study, the diffusion of the peptide CIGB300 in hydrogels of different stiffnesses was evaluated using fluorescence spectroscopy. Soft and stiff silk fibroin (SF) hydrogels were produced, using 4.5 and 9% of PEGDA, respectively, and their Young’s moduli were evaluated using nanoindentation, as shown in Figure 6A, yielding values of 2.29 (±0.05) kPa and 7.57 (±0.09) kPa, respectively.

The peptide was labelled with FITC, and 100 µL of 3 mM of FITC-CIGB300 was deposited on top of an SF hydrogel (900 µL) previously polymerized directly in a cuvette. Although FITC may affect the peptide diffusion rate by modifying the hydrodynamic radius and hydrophobicity, here, the diffusion of the same FITC-labeled peptide in two SF hydrogels with different stiffnesses was compared. The variation in fluorescence emission intensity at λ_em_ = 515 nm was monitored over time. Fluorescence emission of the hydrogels at λ_em_ = 515 nm was assessed before the addition of the peptide solution on top of the hydrogel and subtracted as a background signal. The diffusion of the FITC molecule alone in the stiffer SF hydrogel over time was also assessed, as shown in Figure 6B. The diffusion kinetics of FITC were very fast, showing a hyperbolic trend. In 1 h, the fluorescence emission intensity of FITC was at its maximum and remained almost constant over time, demonstrating that small molecules like FITC are able to rapidly diffuse in SF hydrogels. By contrast, the diffusion rate of FITC-CIGB300 was slow and dependent on the SF hydrogels’ stiffness (Figure 6C and Figure S1). The diffusion of FITC alone is rapid and not significantly hindered in hydrogels with higher rigidity; consequently, the observed differences in diffusion can reasonably be attributed to the peptide moiety of the molecule. The profiles of the diffusion kinetics of FITC-CIGB300 were sigmoidal for both hydrogels, indicating the presence of different steps in the diffusion process, and the Hill coefficients of FITC-CIGB300 were 2.94 and 2.24 in the soft and stiff SFHy, respectively. The calculated diffusion rates were approximately 11.5 and 6.35 μM/h in the soft and stiff SFHy, respectively. This behavior is most likely related to the polymeric phase of the hydrogel and its swelling [38,39], in addition to the interaction of the peptide with the protein component of the hydrogel [40,41,42].

2.6. Tumoroid Stiffness Affects the Drug Resistance of the CAL 27 Cancer Cells

Considering the faster diffusion kinetics of CIGB300 in the soft SF hydrogel, the major cell death observed in the tumoroids at higher stiffnesses could be due to an increased sensitivity to the antitumor peptide of the cells grown under stiffer conditions. Therefore, the antitumor property of the peptide CIGB300 may be strengthened by the physical rheological factor in 3D head-and-neck tumoroid models in a synergistic way. As described above, the antitumor effects of CIGB300 are related to the down-regulation of B23/NPM1, which is an important tumor marker [15,22,43], through physical interaction and the inhibition of the activation by phosphorylation at Ser 125 via casein-kinase 2 (CK2) in the tumor cells [44,45,46,47]. Several studies have shown that B23/NPM1 is over-expressed in solid tumors with different histological origins, including tumors of the pancreas [48], prostate [49], and liver [50]; glioma and glioblastoma [51,52]; astrocytoma [53]; thyroid tumors [54], gastric tumors [55]; colon tumors [56]; and others. Further, NPM1 has been proposed to be an adverse prognostic marker in a number of such malignancies [57,58] due to its overexpression, which is often correlated with high mitotic and metastasization indices. On these bases, B23/NPM1 protein expression under different rheological conditions was assessed in this study. Figure 7A shows the results of Western blot analyses evaluating the expression of B23/NPM1 in CAL27 cultured under both 2D and 3D conditions and at different stiffnesses (CAL27PSF_soft_ and CAL27PSF_stiff_). A significant decrease in B23/NPM1 expression was observed under 3D conditions relative to the 2D culture, indicating that B23/NPM1 expression changed in response to the microenvironment of the cell culture system. However, no significant differences in B23/NPM1 expression were observed between the two stiffnesses (CAL27PSF_soft_ and CAL27PSF_stiff_). Therefore, B23/NPM1 expression in the 3D-CAL27 cell culture system does not change in the 3.4–7.8 kPa stiffness range. We also evaluated P-gp expression in both the soft and stiff tumoroids. P-glycoprotein (P-gp) is an ABC transporter associated with resistance to cancer chemotherapy [59]. Figure 6 shows representative confocal fluorescence micrographs of CALPSF_soft_ and CAL27PSF_stiff_ after 3 days of cell culture. A major expression in P-gp expression was observable in CAL27PSF_soft_ relative to CAL27PSF_stiff_ in the confocal fluorescence analyses, and it was also confirmed by Western blotting analysis (Figure 7B), where a statistically significant difference in P-gp expression at the two different stiffnesses was observed.

Although it is not known if the P-gp protein is involved in the transport of CIGB300 out the cell, some studies indicate that cancers’ resistance to some antitumor cyclic peptides, such as FK228 and apicidin, is related to P-gp expression [60]. These preliminary data could open the way for studying the correlation between the activity of this cyclic antitumor peptide and drug resistance. Moreover, P-gp function is relevant in the endogenous detoxification system from metabolites; therefore, its down-regulation could be extremely important for sensitizing a cell to these drugs. P-gp expression has been also associated with B23/NPM1 expression [61]: the knockdown of B23/NPM1 reduces drug resistance through the down-regulation of P-gp. In agreement, we also observed a decrease in P-gp expression associated with B23/NPM1 down-regulation under stiff conditions relative to cells grown on TCP.

These results indicate relevant effects of stiffness on drug resistance in head and neck tumoroids. Therefore, the increase in the sensitivity of the tumor cells to the CIGB300 peptide treatment may be due to a synergistic effect with the physical rheological factor that induces a change in the cancer cells.

3. Materials and Methods

3.1. Molecular Studies

3.1.1. Fluorescence Spectroscopy

Steady-state fluorescence spectra were measured through a spectrofluorometer, namely, a “Fluoromax-4” (Horiba Jobon Yvon, Edison, NJ, USA), in a thermostated (25 °C) quartz cell (1 cm). The fluorescence emission spectra of W residues in 163 μM CIGB300 aqueous solution (λex = 280 nm, λe**m = 290–450 nm) were measured with both excitation and emission slits set to 2 nm, integrating the signal for 0.6 s at each nanometer. FITC and FITC-CIGB300 fluorescence intensities were measured with λex = 495 nm and λe**m = 515 nm, with excitation and emission slits set to 3 and 5 nm, respectively, integrating the signal for 0.4 s at each nanometer. Each value was the mean of four acquisitions and two experimental replicates. The data were fitted using the following sigmoidal curve equation:

$$[eqn]$$

where ΔF denotes (F_1_ − F_0_); ΔF_max_ denotes (F_max_ − F_0_); t denotes time; t_ΔFmax/2_ denotes time where ΔF is half of the ΔF_max_; and S is the sigmoid Hill coefficient.

3.1.2. Circular Dichroism of CIGB300

Far-UV CD measurements were performed using a Jasco 1500 spectropolarimeter (Jasco, Tokyo, Japan) calibrated with camphorsulfonic acid. CD spectra were recorded between 190 and 260 nm using a path length of 0.1 cm, a time constant of 1.0 s, a bandwidth of 2 nm, a scan rate of 0.2 nm/min, and a sensitivity of 20 mdeg. The CD measurements were performed on 67 μM CIGB300 in aqueous solutions using a quartz cell with an optical length of 0.1 cm thermostated at 25 °C. The spectrum was the mean of three acquisitions and three experimental replicates.

3.2. Rheological Analyses of the Hydrogels

The microrheological analyses of SF and PSF hydrogels at high and low stiffness (without cells) were performed via nanoindentation using PIUMA nano-indenter equipment (Optics11 Life, Amsterdam, The Netherlands). SFHy and PSFHy samples were produced as follows. SF was purified from Bombix mori cocoons and lyophilized through a published protocol, and PSF was obtained through PEGylation of SF, as previously described [62]. The hydrogels were prepared via photopolymerization (5 min, 365 nm, 5 mW/cm^2^) of 100 μL of the gel precursor solutions (3 mg/mL of SF or 70 µL of 6 mg/mL PSF in PBS buffer, 30 µL (stiff) or 15 µL (soft) of 30% w/v PEGDa 10 kDa, and 1% w/v of Irgacure 2959 for a total volume of 100 µL in PBS), using o-rings as molds. The samples were then analyzed in liquid environment using PBS buffer. A probe with a cantilever stiffness of 0.45 N/m and a spherical tip with a 49.5 µm radius was selected for the measurements. We measured the effective Young’s Modulus (E_eff_, Pa) in peak load poking mode, performing a matrix scan of 6 × 6 and 4 × 4 with a displacement on the X and Y axes of 20 μm (max load, 0.6–1.40 µN; piezo speed, 30.0 µm/s) in order to mediate the E_eff_ value of the gels. Data analysis was performed by establishing a Hertzian contact model and obtaining a 3D plot of E_eff_ values across the matrix. Along with the matrix scan, one representative load–indentation curve is reported for each sample analyzed.

3.3. Cellular Studies

3.3.1. Cell Culture System Production and Cell Viability Assay

Cell studies were performed using human head and neck squamous cell carcinoma (HNSCC) cells, specifically adherent cells of the CAL27 cell line (Istituto Zooprofilattico, Brescia, Italy). Cell cultures were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Milan, Italy), containing 10% v/v Fetal Bovine Serum (FBS) (Gibco, Italy), 1% w/v penicillin–streptomycin (Sigma-Aldrich, Milan, Italy), 1% w/v of a 200 mM L-Glutamine solution (Gibco, Italy) and 1% v/v non-essential amino acid solution (Sigma-Aldrich, Italy). CAL27PSF hydrogel spheres were obtained using the solution precursor of PSF (for a final volume of 100 µL in PBS buffer: 70 µL of PSF 6 mg/mL dissolved in PBS; 30 µL or 15 µL of PEGDa 10 kDa 30% w/v for stiff or soft, respectively; and 1% w/v of the photo-initiator Irgacure 2959). Using the procedure reported by Patent LDO0252, a polydimethylsiloxane (PDMS)-based superhydrophobic nanostructured surface was prepared [4,5,63]. CAL27 cells at a density of 10^4^ cells/µL were resuspended in the gel precursor solution, deposited (drops of 3–5 µL) on the superhydrophobic surface, and photopolymerized by exposure to UV light (365 nm, 5 mW/cm^2^) for 2 min. 3D cell systems were cultured and monitored over time via brightfield microscopy using a Zeiss microscope (Primovert, Zeiss, Milan, Italy). The peptide CIGB300 was diluted in Dulbecco’s Modified Eagle Medium (DMEM) without FBS at 100 mM, aliquoted and stored at −20 °C, and used for the treatments at the different concentrations.

WST-1 (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolium]-1,3-benzene disul-fonate) assay (Cell Proliferation Reagent WST-1, Roche, Mannheim, Germany) [64] was performed to assess cell viability and proliferation after each cell treatment with CIGB300. CAL27 cells were seeded at 1.6 × 10^4^ cells/cm^2^ in a 96 w. multiwell plate and, on the next day, treated for 24 h with CIGB300 (80, 160, 240 and 320 μM). 3D CAL27PSF spheroids were produced as described above and, on the day after their production, treated with CIGB300 (80, 160, 240 and 320 μM) for 72 h. After each treatment, the medium was replaced with fresh DMEM high-glucose medium without phenol-red (GIBCO, Italy) containing tetrazolium salt WST-1 (5% v/v). 2D-seeded cells and 3D cell samples were then incubated for 2 h and 4 h, respectively, at 37 °C and 5% CO_2_. Absorbance of the medium was recorded at 450 nm using a microplate reader (iMark™ Microplate Absorbance Reader, Bio-Rad, Milan, Italy). The cell viability of the CAL27PSF systems was also assessed with LIVE/DEAD^®^ Cell Imaging Kit (L/D assay) (488/570) (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Milan, Italy) using a Zeiss Axio Observer 7 fluorescence microscope (Zeiss, Milan, Italy).

3.3.2. Immunofluorescence Microscopy Analyses

Fluorescence analysis of CAL27PSFs was performed by first staining the nuclei of live cells with Hoechst 33,342 (Sigma-Aldrich, Italy) for 4 h. Following staining, spheres were rinsed with PBS, fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 30 min, and analyzed using a Zeiss Axio Observer 7 microscope. For immunofluorescence analysis, spheres were similarly stained with Hoechst 33,342 (Sigma-Aldrich, Italy) and then fixed in 4% PFA at room temperature for 30 min. Samples were permeabilized for 15 min with a 0.3% Triton X-100 solution in PBS and incubated overnight at 4 °C in a blocking buffer containing 10% BSA (w/v), 0.1% Triton X-100 (v/v), and 1% glycine (w/v) in PBS. After blocking, CAL27PSFs were incubated overnight with 1% BSA and 20 mM Gly solution in PBS containing the primary antibody for P-gp (rabbit) (Abcam, Cambridge, UK) followed by the appropriate Alexa fluorochrome-conjugated secondary antibody (488 nm, green) (Thermo Fisher Scientific, Invitrogen, Carlsbad, CA, USA). Confocal microscopy was performed using a Stellaris Leica microscope platform.

3.3.3. Scratch Wound-Healing Assay

CAL27 cells were seeded into a 24-well plate (6.5 × 10^4^ cells/cm^2^) and cultured at 37 °C with 5% CO_2_ so that the cells would reach confluency the next day. A scratch wound was created, and the area of the scratch-wound was measured at time 0 and after 24 h. Percentage of wound closure was measured as follows:

$$[eqn]$$

3.3.4. Evaluating Protein Expression via Western Blot Analysis

Proteins were extracted from CAL27 cells and CALPSF spheres by lysing the samples in 100 μL of SDS-PAGE sample buffer. Cell samples were then vortexed, boiled for 5 min, centrifuged at 10,000 rpm for 5 min, and stored at −20 °C. Protein extracts were separated on 12 or 15% polyacrylamide gel and transferred onto PVDF membrane (Sigma-Aldrich, Italy). Membranes were blocked and incubated overnight at 4 °C with the following primary monoclonal antibodies: Ab-Acetyl-Histone H3 [Ac-Lys9] (rabbit) (Sigma-Aldrich, Italy), Ab-CyclinD1 (rabbit) (Cell Signaling Technology, Danvers, MA, USA), Ab-B23/NPM1 (rabbit) (Merck, Sigma Aldrich, Italy) or Ab-P-gp (rabbit) (Abcam, Cambridge, UK). Secondary-antibody (Cell Signaling Technology, USA) incubation (dilution 1:3000) was then performed for 4 h at room temperature. To control for protein loading, immunoblots were also probed with Ab-β-tubulin mouse (Sigma-Aldrich, Milan Italy). Protein signals were visualized using the Super Signal West Pico kit (Thermo Scientific, Waltham, MA, USA) and detected with a Fluorchem Imaging system (Alpha Innotech Corporation-Analitica De Mori, Milan, Italy). Quantitative analysis of protein expression in CAL27 tumoroids was performed using 5 microspheres per sample, providing an inherently mediated value.

3.3.5. Statistical Analysis

The statistical analysis was performed using GraphPad Prism version 8.0 for Windows (GraphPad Software, San Diego, CA, USA). Data from three to six biological replicates were quantified and analyzed for each variable using a one-tailed Student’s t-test or one-way ANOVA test followed by Dunnett’s multiple-comparisons test. A p value of <0.05 was considered to be statistically significant. Standard deviations or standard error means were calculated and presented for each experiment.

4. Conclusions

Head–neck tumoroids were produced and characterized for in vitro studies of the antitumor activity of the peptide CIGB300, with the mechanical properties of the cellular microenvironment taken into account. A spectroscopical characterization of CIGB300 was performed, revealing an absence of peptide aggregation in the concentration range used for the cell treatment, although it is not possible to totally exclude aggregation processes due to the presence of other components, such as cells and hydrogel. Inhibition of the cell viability and migration of oral squamous cell carcinoma cell line CAL27 were observed in a 2D cell culture system, a finding that is in agreement with previous studies on different cancer cell lines. The antitumor effects were also validated using 3D CAL27 culture systems. Tunable CAL27PSF tumoroids were produced and characterized, and the antitumor activity of CIGB300 was also assessed in relation to the mechanical properties change of the cellular microenvironment. The major antitumor effect was observed using a stiffer microenvironment. The diffusion of CIGB300 over time was investigated on silk fibroin hydrogels at the same Young’s modulus via fluorescence spectroscopy. A cooperative diffusion behavior of the peptide for both the hydrogels at different stiffnesses was observed, a finding that is in agreement with previous studies on the diffusion of molecules in hydrogels [38,39]. Significant differences in the diffusion rate of the peptide in the two hydrogels were observed, which were in agreement with their Young’s moduli. Our results demonstrate an effect of stiffness on the increase in the sensitivity of the tumor cells to CIGB300 treatment, which was independent of the diffusion rate in the system of the peptide in the microenvironment. B23/NPM1 and P-gp expression, either 2D versus 3D or soft versus stiff, were assessed, revealing only a significant decrease in B23/NPM1 expression for 2D versus 3D under the rheological conditions used but a significant decrease in P-gp expression for soft versus stiff, suggesting there is a correlation between cyclic antitumor peptides’ activity and drug resistance. In conclusion, these results suggest that the increase in the sensitivity of the CAL27 cells to the CIGB300 treatment may be due to a synergistic effect with respect to the physical rheological factor that induces a change in the drug resistance of the cancer cells, indicating microenvironment rheology should be targeted for optimizing anticancer therapies. Further studies using different cell lines, a wider range of stiffnesses, and in vivo models are needed to help us fully understand how the mechano-physical properties of the cellular microenvironment may affect peptide-based drugs sensitivity of tumors.