3D-printed scaffold harboring copper ions combined with near-infrared irradiation for local therapy of cancer

Summary

Cancer is a major health threat and a leading cause of human death worldwide. Surgical resection is the primary treatment for most cancers; however, some patients develop locoregional recurrence. Here, we developed an in situ cancer therapeutic system aimed to locally treat cancer and prevent postoperative recurrence. A functional scaffold, based on alginate/gelatin and crosslinked with copper ions, was fabricated by 3D printing and showed an excellent photothermal effect under near-infrared (NIR) irradiation. The combination of copper ions and NIR effectively killed thyroid cancer cells and patient-derived organoids, indicating a synergetic and broad-spectrum antitumor effect on thyroid cancer through the chemo-photothermal therapy. This implantable stent is designed to provide effective treatment in the vicinity of the tumor site and can be degraded without secondary surgery. The copper-loaded hydrogel scaffold may be a potential candidate for local cancer treatment and pave the way for precise and effective cancer therapy.

Graphical abstract

Highlights

• •3D printing a copper-loaded scaffold for local cancer therapy under NIR irradiation
• •Establishment and characterization of thyroid cancer organoids
• •The scaffold exhibits a chemo-photothermal therapeutic effect on thyroid cancer

Biomaterials; Biomedical Engineering; Cancer; Materials science

Introduction

Cancer is one of the main causes of human death worldwide, and its incidence is increasing year by year. Surgical resection, which aims to remove tumor tissue directly, is the main treatment option for cancer patients. However, local tumor recurrence resulting from minimal or occult residual tumors remains a serious clinical problem. Postoperative adjuvant therapies, such as targeted therapy and systemic chemotherapy, can improve the removal of residual tumor cells and prevent their recurrence and metastasis. Yet, these treatments still face problems of severe toxicity and non-specific distribution to normal cells and tissues. Thus, developing new treatment strategies to overcome these limitations has become a hot topic in cancer research.

In recent years, some novel and effective strategies for cancer treatment have received great attention. As a non-invasive and spatiotemporal controlled treatment technology, photothermal therapy (PTT) has gained a significant role in clinical practice. Upon irradiation with near-infrared (NIR) laser, the heat generated from the absorbed optical energy by photothermal agents can destroy cancer cells through hyperthermia.¹ PTT can reduce tumor burden through local thermal ablation, rendering it a powerful tool for cancer treatment.² By locally irradiating with NIR, the hyperthermia (>42°C) generated by photothermal agents can induce apoptosis or necrosis of tumor cells by inhibiting DNA synthesis, damaging cytoskeleton, and destroying cell membrane.^3,4 Chemodynamic therapy (CDT), which converts overexpressed endogenous hydrogen peroxide (H₂O₂) to strong oxidized hydroxyl radicals by catalyzing metal ion-mediated Fenton or Fenton-like reactions, plays a role in tumor therapy.^5,6 In CDT, the level of reactive oxygen species (ROS) generation plays an important role in evaluating treatment efficacy. Copper ions-based materials, which function as photosensitizers or Fenton-reaction catalysts, are capable of generating ROS. The generation of ROS can further contribute to the elimination of cancer cells. Moreover, PTT can improve several ROS-related therapy strategies such as photodynamic therapy (PDT) and CDT in different ways.⁷ Therefore, integrating Cu²⁺-based PTT and ROS-related therapy into a single platform appears to be an ideal strategy for synergistic cancer treatment. Copper ions have been extensively studied for their photothermal and chemodynamic effects in cancer therapy.^{8,9,10,11,12,13} The traditional method primarily carries metal ions based on nanomaterials. However, the uncertain structure of nanomaterials may result in uncontrolled catalytic effects on Fenton or Fenton-like reaction systems and still cannot change the status quo of systemic delivery.¹⁴

Relevant studies have developed various delivery systems for therapeutic methods, such as hydrogels, polymer scaffolds, polymer micelles, hydrogel/polymer micelle composites, and stimulation-responsive materials.¹⁵ However, these vectors have certain shortcomings as they need to be injected into the body to reach the lesions; especially they are rapidly cleared in the blood circulation and then excessively accumulate in non-target tissues.¹⁶ Local delivery using a 3D-printed stent is an effective method to treat local tumor residue and recurrence, compared to injecting drugs into the systemic circulation. This method minimizes drug distribution throughout the body thereby reducing side effects. Therefore, local delivery using a 3D-printed stent will be a promising way for cancer patients with residual disease and local recurrence.

In this paper, we propose a simple strategy, an implant called Cu/AG scaffold, for cancer treatment. It is based on alginate/gelatin (AG) and crosslinked with cupric chloride (CuCl₂). The localized release of copper ions ensures the efficacy of CDT at the lesions, while the photothermal effect of copper ions further reduces the use of copper ions and minimizes potential side effects. We validated the therapeutic effect of the scaffold on six thyroid cancer cell lines and conducted further experiments using six patient-derived organoid cultures derived from clinical tumor specimens. These results demonstrated that the method has a broad-spectrum therapeutic effect in the treatment of thyroid cancer. This proof-of-concept study presents a promising way to develop functional stents for the treatment of residual tumors and prevent postoperative recurrence of cancer.

Results and discussion

Fabrication and characterization of Cu/AG scaffolds

As the most widely used bioink in 3D bioprinting, alginate was chosen as the main material for the stent due to its excellent biocompatibility and the obvious advantages of easy gelatinization using divalent cationic crosslinking under mild conditions.¹⁷ Gelatin was added for its properties of good biocompatibility and printability, as well as thermosensitive sol-gel transition.^18,19 In our previous studies, we reported a range of concentrated bioinks using alginate and gelatin.^20,21,22 These concentrated bioinks showed good printability, enhanced mechanical characteristics, and robust structural stability. The printing ink based on alginate undergoes a transition from sol to gel through crosslinking by copper ions (Figure S1). By crosslinking with CuCl₂, the scaffold introduces copper ions into the alginate matrix, allowing it with both chemodynamic and photothermal effects against tumors. The amount of copper ions carried by the stent can be controlled by adjusting the concentration of the crosslinking agent to achieve an accurate therapeutic effect. This 3D-printed scaffold can be surgically implanted into the human body; its structure and size can be customized by computer-aided design to perfectly match the surgically induced tissue defects. Due to the degradability of the materials used, surgical removal is not required after treatment, and the material can be degraded by itself.

Here, the Cu/AG scaffolds with different concentrations of copper ions were fabricated through an extrusion-based 3D printing technology by crosslinking with different concentrations of CuCl₂ (0, 0.07, 0.1, 0.13, and 0.16 mol/L), denoted as 0-Cu/AG, 0.07-Cu/AG, 0.1-Cu/AG, 0.13-Cu/AG, and 0.16-Cu/AG, respectively. Optical images showed that the color of the resultant scaffolds became darker with the increase of the incorporated copper ions (Figure 1A). The scaffolds exhibited smooth surfaces and uniform structures as observed by scanning electron microscope (SEM) (Figure S2). Energy-dispersive X-ray spectroscopy (EDS) element mapping analysis showed that these elements were evenly distributed in the scaffold (Figure 1B), confirming the successful encapsulation of Cu²⁺. Atomic percentage analysis revealed an increased percentage of copper atoms on the surface of the scaffold with the increasing of the concentration of crosslinking agent (Figure 1C). The measurement of mechanical properties and Young’s modulus indicate that the stiffness of the Cu/AG scaffolds is dependent on the concentration of Cu²⁺ in the crosslinking agent (Figures 1D and 1E). The release of Cu²⁺ lasted for about 4 days, and the release rate gradually slowed down compared to the initial burst release (Figure 1F). The Cu/AG scaffolds with a high concentration of crosslinking agent exhibited much higher ionic release (Figure 1F). The in vitro degradation of the Cu/AG scaffolds was examined by incubating them in PBS (pH = 7.4, 37°C). As the crosslinking density of Cu²⁺ increased, the degradation rate of the AG scaffold decreased (Figure 1G). Moreover, the degradation profile of the 0.13-Cu/AG scaffolds was simulated in both the normal physiological environment (pH 7.4) and the tumor microenvironment (pH 6.5). There was no significant impact on the degradation of the scaffolds in pH 6.5 and pH 7.4 in the first 5 days. After 5 days, the scaffolds in both the pH 6.5 and pH 7.4 environments exhibited accelerated degradation, which was noticeably different from the degradation rate in a neutral environment (Figure 1H).

Figure 1.

Fabrication and characterization of Cu/AG scaffolds

(A) Optical images of the Cu/AG scaffolds crosslinked with 0, 0.07, 0.1, 0.13, and 0.16 mol/L CuCl₂, respectively. Scale bar, 6 mm.

(B) Energy-dispersive X-ray spectroscopy (EDS) elemental mapping analysis of the 0.13-Cu/AG scaffold. Scale bar, 1 mm. See also Figure S2.

(C) Atomic percentage on the surface of Cu/AG scaffolds with different concentration of the crosslinking agent. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

(D) The Young’s modulus of the Cu/AG scaffolds with different concentration of the crosslinking agent. ∗p < 0.05.

(E) The compressive strength of the Cu/AG scaffolds with different concentration of the crosslinking agent. ∗p < 0.05, ∗∗p < 0.01.

(F) In vitro release of copper ions from Cu/AG scaffolds with different concentration of the crosslinking agent.

(G) In vitro degradation of Cu/AG scaffolds with different concentration of the crosslinking agent.

(H) In vitro degradation of 0.13-Cu/AG scaffolds with different pH values.

(I) Photothermal heating curves of different Cu/AG scaffolds under NIR laser irradiation (1.3 W/cm²).

(J) Photothermal heating curves of the 0.13-Cu/AG scaffolds under NIR laser irradiation at different laser power (0.7, 1.0, 1.3, and 1.6 W/cm²). See also Figure S4.

(K) Photothermal stability of the 0.13-Cu/AG scaffolds under sequential laser on/off cycles. See also Figure S5.

Furthermore, we assessed the in vitro photothermal performance of the 3D-printed Cu/AG scaffolds. The 808 nm NIR light was chosen for the photothermal application with the Cu/AG scaffold due to its highest absorption rate in the UV-vis-NIR absorption spectra (Figure S3). Under NIR laser irradiation (1.3 W/cm²) for 2.5 min, a significant temperature rise in Cu/AG scaffolds was observed with a dependence on the Cu²⁺ concentration (Figure 1I). By comparison, the temperature of the AG scaffold only increased by ∼8.5°C under the same irradiation conditions. The photothermal effects of the Cu/AG scaffolds were dependent on the laser power density. As the laser power increased, the temperature of the Cu/AG scaffolds also elevated (Figures 1J and S4). Additionally, no significant temperature decrease was found even under five cycles of laser irradiation (Figures 1K and S5).

Copper ions, as a photothermal agent in PTT, have the important characteristic of photothermal conversion. Therefore, the photothermal conversion efficiency of Cu/AG scaffold was evaluated. The photothermal conversion efficiency (η) of the 0.13-Cu/AG scaffold can be calculated according to the following equation:

$$\eta =\frac{hA\Delta T_{\max }-Qs}{I\left(1-{10}^{-A\lambda }\right)}$$

where h is the heat transfer coefficient, A is the surface area of the container, ΔT_max is the temperature change of the 0.13-Cu/AG scaffold at the maximum steady-state temperature, Q_s is the heat associated with the light absorbed by solvent, I is the laser power, and A_λ is the absorbance of the 0.13-Cu/AG scaffold at the wavelength of 808 nm. According to the equation, the η value of 0.13-Cu/AG scaffold was calculated to be about 36.51%. The high photothermal conversion efficiency of the Cu/AG scaffold makes it a promising agent for PTT.

Treatment of thyroid cancer cells using fiber scaffolds

Six thyroid cancer cell lines, BCPAP, 8305C, CAL-62, FTC-133, K1, and TPC-1, were used to evaluate the therapeutic effect of the Cu/AG stent on thyroid cancer. Cell viability of BCPAP cells in the 0.13-Cu/AG + NIR scaffold group decreased by 69.88%, while the 0.13-Cu/AG scaffold group showed a limited decrease of 29.7% (Figure 2A). Cell viability in the AG + NIR group remained over 94%. In contrast, AG scaffold has no significant effect on cell growth, indicating that the AG scaffold has non-cytotoxicity (Figure 2A). Live/dead staining of BCPAP cells in different treatment groups showed that the 0.13-Cu/AG + NIR (1.3 W/cm²) scaffold induced significant and extensive cell death compared with other groups (Figure 2B). Subsequently, concentration gradient of Cu²⁺ experiments was performed on the six thyroid cancer cell lines. Under NIR laser irradiation (1.3 W/cm²), the 0.13-Cu/AG and 0.16-Cu/AG scaffolds triggered >70% cell ablation, which were far superior to the other groups (Figures 2C, 2D, and S6). Furthermore, cell viability measurements after treating the six thyroid cancer cell lines with 0.13-Cu/AG + NIR revealed that the 3D-printed copper-loaded scaffold had a broad-spectrum therapeutic effect on thyroid cancer cells (Figure 2E).

Figure 2.

Treatment of thyroid cancer cells with fiber scaffolds

(A) Cell viability of BCPAP cells in different treatment groups. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

(B) Live and dead staining of BCPAP cells in different treatment groups. The concentration of Cu²⁺ is 0.13 mol/L. Scale bar, 200 μm.

(C) Cell viability of BCPAP cells in the Cu/AG + NIR group with different capacities loaded of Cu²⁺. ∗p < 0.05, ∗∗p < 0.01. See also Figure S6.

(D) Cell viability of CAL-62 cells in the Cu/AG + NIR group with different capacities loaded of Cu²⁺. ∗p < 0.05, ∗∗p < 0.01. See also Figure S6.

(E) Cell viability of six thyroid cancer cell lines after treatment with 0.13-Cu/AG + NIR.

(F) The fluorescence intensity change curves of ROS production in BCPAP cells of different treatment groups.

(G) The fluorescence intensity change curves of ROS production in CAL-62 cells of different treatment groups.

CDT is a promising approach for cancer treatment, which utilizes Fenton or Fenton-like reactions to generate toxic hydroxyl radicals, disrupt cellular redox homeostasis, and induce cell death. Several studies have shown that Cu²⁺ can increase intracellular levels of ROS through Fenton-like reactions, leading to cell damage or death.^9,10,11 In this study, we examined the production of ROS in different treated groups. As shown in Figures 2F and 2G, the Cu/AG scaffold group exhibited the generation of ROS in BCPAP and CAL-62 cells, while the control, AG, and AG + NIR groups showed no ROS production. Additionally, the Cu/AG + NIR scaffold also induced ROS production, indicating that PDT induced by NIR may also be involved.

Collectively, these experiments suggest that the combination of CDT (Cu²⁺) and PTT (heat) exhibits a synergistic effect on thyroid cancer cell lines, probably due to the enhanced cytotoxicity of Cu²⁺ in the elevated temperature of the Cu/AG stent.

Establishment and histological characterization of thyroid cancer organoid models

In the last decade, patient-derived organoids have facilitated basic cancer research and played an important role in personalized medicine. By developing organoids from human tumor tissues, they have opened new avenues for studying and treating various cancers. Tumor organoids have been utilized for generating biobanks, performing drug screening, and studying mutational signatures. In contrast to immortalized cancer cell lines that often cannot fully preserve intratumor heterogeneity and cellular diversity,^23,24 tumor organoids have been used as ex vivo models that faithfully recapitulate histological features, genetic alterations, and tumor heterogeneity of the original tumors. Animal models, particularly patient-derived mouse xenografts (PDXs), have provided significant insights into the cellular and genetic basis of tumors.²⁵ However, they are often costly, inefficient, time-consuming, and difficult to translate into clinical treatment.^26,27 Organoids have been used to model various types of cancer and can accurately predict patient responses to drugs and radiotherapy in the clinic.

In our study, six thyroid cancer organoid models were successfully established from surgically removed thyroid tumors (Figure 3A). The histological features of thyroid cancer organoids were compared with those of the parental tumors using H&E staining. Thyroid cancer organoids exhibited similar histological characteristics to those of the original tumors, including growth patterns and cellular and nuclear atypia (Figure 3B). Next, we assessed the expression of a panel of thyroid cancer biomarkers, cytokeratin 19 (CK19), galectin-3, and Ki-67, in tumor tissue-organoid pairs. Immunofluorescence staining demonstrated that the marker expression profile of each thyroid cancer organoid line matched that of its corresponding parental tumor (Figure 4). These results suggest that the histological characteristics and marker expression of the original tumors were well preserved in the derived organoid models.

Figure 3.

Establishment and histological characteristics of thyroid cancer organoids

(A) Representative bright-field microscopy images of thyroid cancer organoid lines. The passage number for each patient’s organoid line was: patient 1, P3; patient 2, P2; patient 3, P4; patient 4, P5; patient 5, P4; patient 6, P3. Scale bar, 100 μm.

(B) H&E staining of thyroid cancer tissues and the derived organoids. Scale bar, 100 μm.

Figure 4.

Immunofluorescence staining of CK19, gal-3, and Ki-67 in the primary tumor tissues and the paired organoids

The passage number for each patient’s organoid line was: patient 1, P3; patient 2, P2; patient 3, P4; patient 4, P5; patient 5, P4; patient 6, P3. T, tumor; O, organoids; gal-3, galectin-3. Scale bar, 100 μm.

Treatment of thyroid cancer organoids using fiber scaffolds

To further confirm the therapeutic effect of the Cu/AG scaffolds on thyroid cancer, we used them to treat thyroid cancer organoids derived from six patients under NIR light. For instance, organoid viability of patient 1 in the 0.13-Cu/AG and 0.13-Cu/AG + NIR (1.3 W/cm²) scaffold groups decreased by 68.7% and 95%, respectively (Figure 5A). Light microscopy imaging of thyroid cancer organoids derived from patient 1 and 3 showed that treatment of organoids with 0.13-Cu/AG + NIR resulted in an altered phenotypic appearance of organoids (Figure 5B). Furthermore, calcein-AM/PI staining indicated that Cu/AG+NIR-treated organoids from patient 1 (Figure 5C) and patient 3 (Figure 5D) exhibited significant and extensive cell death compared to the control group. The combination of CDT and PTT of Cu/AG stent showed a great therapeutic effect on thyroid cancer organoids derived from different patients, indicating a broad-spectrum antitumor effect in patients with thyroid cancer (Figure 5E).

Figure 5.

Treatment of thyroid cancer organoids with fiber scaffolds

(A) Cell viability of thyroid cancer organoids derived from patient 1 in different treatment groups. The concentration of Cu²⁺ is 0.13 mol/L. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

(B) Representative bright-field images of thyroid cancer organoids in the control and 0.13-Cu/AG+NIR groups. Scale bar = 100 μm except the insets were 50 μm.

(C) Live and dead staining of thyroid cancer organoids derived from patient 1 in the control and 0.13-Cu/AG+NIR groups. Scale bar, 50 μm.

(D) Live and dead staining of thyroid cancer organoids derived from patient 3 in the control and 0.13-Cu/AG+NIR groups. Scale bar, 50 μm.

(E) Cell viability of thyroid cancer organoid lines derived from six patients after treatment with 0.13-Cu/AG + NIR.

The Cu/AG scaffold with NIR that we developed provides a promising therapeutic platform for localized treatment of thyroid cancer. Cu²⁺ can simultaneously serve as a photothermal agent to mediate PTT and as a catalyst in Fenton-like reactions to generate ROS. On this platform, minimizing negative effects can be achieved by controlling the local concentration of Cu²⁺ and the power of NIR. The combination of dual therapies can reduce the use of Cu²⁺ and mitigate potential side effects. Our previous study has shown that low dose of Cu²⁺ crosslinked scaffolds not only have minimal adverse effects on normal tissues but also promote tissue repair to some extent.²⁸ Furthermore, using low-density laser irradiation and shortening the irradiation time can significantly reduce side effects on normal cells and tissues. In this study, the Cu/AG scaffolds were manufactured through 3D printing, enabling customized shaping and structural design according to individual patient needs. The scaffolds can be implanted into the lesion site after surgery to prevent postoperative recurrence. The scaffold material exhibits high biocompatibility and rapid degradation rate and eliminates the secondary surgical removal after implantation.

Conclusions

In this study, the functional Cu/AG hydrogel scaffolds with chemo-photothermal effect were prepared by 3D printing technology. The Cu²⁺-loaded hydrogel scaffolds exhibited in vitro release dynamics and demonstrated excellent photothermal effect under NIR irradiation. The combination of copper ions and NIR therapy effectively killed thyroid cancer cells as well as thyroid cancer organoids derived from clinical samples. Notably, Cu²⁺-based PTT and CDT combined treatment strategy not only avoids the limitations of single mode of cancer treatment but also optimizes the treatment effectiveness. Therefore, we believe that the 3D-printed hydrogel stent loaded with copper ions may be a potential candidate for local cancer treatment.

Limitations of the study

In this paper, we demonstrated the significant inhibitory effect of the 3D-printed scaffold on the thyroid cancer cell lines and patient-derived organoids. The ongoing study to develop PDX models using thyroid cancer organoids will be more helpful in studying the therapeutic effects of 3D bioprinting scaffolds in in vivo experiments.

In addition to the PTT of the Cu²⁺-loaded hydrogel scaffold combined with NIR, PDT induced by NIR may also be involved. However, the underlying mechanisms are still not well understood and require further study.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Galectin-3	Abcam	Cat#ab76245; Clone: EP2775Y; RRID: AB_2265782
Ki-67	Abcam	Cat#ab16667; Clone: SP6; RRID: AB_302459
Cytokeratin 19 (CK19)	Affinity Biosciences	Cat#AF5106; RRID: AB_2837592
Cy3-labeled goat anti-rabbit IgG (H + L)	Beyotime	Cat#A0516; RRID: AB_2893015
Biological samples
Human papillary thyroid cancer tissues	This study	N/A
Chemicals, peptides, and recombinant proteins
B27	Gibco	Cat#17504-044
N-acetyl-L-cysteine	Sigma-Aldrich	Cat#A9165
Nicotinamide	Sigma-Aldrich	Cat#N0636
R-spondin-1	Peprotech	Cat#120-38
Noggin	Peprotech	Cat#120-10C
SB202190	Sigma-Aldrich	Cat#S7067
A83-01	Sigma-Aldrich	Cat#SML0788
EGF	Peprotech	Cat#AF-100-15
FGF-7	Peprotech	Cat#100-19
FGF-10	Peprotech	Cat#100-26
Y-27632 dihydrochloride	Abmole	Cat#M1817
Sodium alginate	Sigma-Aldrich	Cat#71238
Gelatin	Sigma-Aldrich	Cat#73865
Calcium chloride	Macklin	Cat#C805225
Cupric chloride	Macklin	Cat#C805298
Critical commercial assays
CellTiter-Glo 3D Reagent	Promega	Cat#G9683
Cell Counting Kit-8	MedChemExpress	Cat#HY-K0301
Reactive Oxygen Species Assay Kit	Beyotime	Cat# S0033S
Calcein-AM	Biolegend	Cat#425201
Propidium iodide	Biolegend	Cat#421301
Experimental models: Cell lines
BCPAP	Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China	Cat#SCSP-543
8305C	Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China	Cat#SCSP-540
CAL-62	Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China	Cat#SCSP-546
K1	iCell Bioscience Inc., Shanghai, China	Cat#iCell-h394
FTC-133	Cobioer Biosciences Co., Ltd., Nanjing, China	Cat#CBP61109
TPC-1	Cobioer Biosciences Co., Ltd., Nanjing, China	Cat#CBP60257
Experimental models: Organisms/strains
Human papillary thyroid cancer organoids	This study	N/A
Software and algorithms
Prism GraphPad 7.0	GraphPad Software, Inc., CA, USA	N/A
SPSS 19.0	SPSS, Inc., IL, USA	N/A
OriginPro 8.0	OriginLab Corporation, Northampton, MA, USA	N/A
CorelDraw X7	Corel Corporation, Ottawa, Canada	N/A

Resource availability

Lead contact

Further information and requests for reagents and resources should be directed to and will be fulfilled by the lead contact, Prof. Yongsheng Zhao (zhaoys69@126.com).

Materials availability

The study did not generate new unique reagents and there are no restrictions to availability.

Experimental model and study participant Details

Cell lines

Human thyroid cancer cell lines BCPAP, 8305C, and CAL-62 with STR verification were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Human thyroid cancer cell lines TPC-1 and FTC-133 with STR verification were purchased from Cobioer Biosciences Co., Ltd. (Nanjing, China), and K1 was purchased from iCell Bioscience Inc. (Shanghai, China). All cell lines were routinely cultured in RPMI 1640 or DMEM medium (VivaCell, Shanghai, China) supplemented with 10% fetal bovine serum (10099-141, Gibco), 1% sodium pyruvate (11360-070, Gibco) and 1% non-essential amino acids (11140-050, Gibco) at 37°C with 5% CO₂.

Human subjects and sample collection

Human thyroid cancer tissues were obtained from six patients (four females, 15–43 years old; two males, 28 and 40 years old) at Peking University Shenzhen Hospital, Shenzhen, China. The detailed clinical information of patients is described in Table S1.

The study was approved by the Human Ethical Committee of Peking University Shenzhen Hospital (approval number: 2022-147). The design of this study is in accordance with the Declaration of Helsinki. All patients provided written informed consent before sample collection.

The resected tumor tissues were transferred to ice-cold adDMEM/F12⁺⁺⁺ medium [advanced DMEM/F12 medium (12634-010, Gibco) containing 10 mM HEPES (15630-080, Gibco), 1 × GlutaMAX (3505-0061, Gibco), and 1% antibiotic-antimycotic (15240-062, Gibco)] and shipped to the laboratory on ice for immediate further processing.

Method Details

Materials

Gelatin from porcine skin and sodium alginate from brown algae were obtained from Sigma-Aldrich. Calcium chloride (CaCl₂) and cupric chloride (CuCl₂) were purchased from Macklin (Shanghai, China).

Preparation and characterization of fiber scaffolds

The alginate/gelatin-based printing inks were prepared by mixing 2 g of alginate powder with 10 g of prepared 9 wt % gelatin solution. After the inks were thoroughly mixed, they were loaded into the printing tube equipped with an 18 G nozzle and a 3D printing system (Bioscaffolder 3.1 from Gesim, Germany) was used to produce the hydrogel scaffolds. The printing speed was set at 3.5 mm/s with a dosing pressure of 5–6 bar, and the temperature was kept at 60°C. After fabricating the scaffolds, crosslink the fiber scaffolds with CuCl₂ (0.07, 0.1, 0.13 and 0.16 mol/L) for 3 min, and then crosslink with 1 mol/L CaCl₂ for 1 min. The morphology of the prepared scaffolds was observed by a stereoscopic microscope (TOKYO 163–0914, Japan). The microstructure and elements of the scaffolds were characterized using scanning electron microscopy (SEM, FEI APREO S, Thermo Scientific, Netherland) and energy-dispersive X-ray spectroscopy (EDS). The photothermal performance of the scaffolds was evaluated by 808 nm NIR laser with different power densities (0.7–1.6 W/cm²). The compressive strength of the Cu/AG scaffold (8 × 8 × 6 mm) was measured by a universal testing machine (HZ1007C, LI XIAN YI QI, China) at a cross-head rate of 5 mm/min. The modulus was taken from the slope of the liner segment of the strain-stress curve.

Photothermal conversion efficiency of Cu/AG scaffold

Photothermal conversion efficiency (η) of the 0.13-Cu/AG scaffold was calculated according to the methods reported previously.^29,30,31,32 The η value can be calculated according to the following equation:

$$\eta =\frac{hA\Delta T_{\max }-Qs}{I\left(1-{10}^{-A\lambda }\right)}$$

where h is the heat transfer coefficient, A is the surface area of the container, ΔT_max is the temperature change of the 0.13-Cu/AG scaffold at the maximum steady-state temperature, Q_s is the heat associated with the light absorbed by solvent, I is the laser power, and A_λ is the absorbance of the 0.13-Cu/AG scaffold at the wavelength of 808 nm. Details of the calculation process are given as following:

The total energy balance of this system as following equation:

$$\sum _i{m}_i{C}_{p,i}\frac{dT}{dt}=Q_{\text{Cu}/\text{AG}}+Qs-Q_{loss}$$ (Equation 1)

where m and C_p are the mass and heat capacity, respectively. T is the solution temperature. Q_Cu/AG is the photothermal energy absorbed by the 0.13-Cu/AG scaffold:

$$Q_{\text{Cu}/\text{AG}}=I\left(1-{10}^{-A\lambda }\right)\eta$$ (Equation 2)

where I is the laser power, A_λ is the absorbance of the 0.13-Cu/AG scaffold at 808 nm, and η is the photothermal conversion efficiency of the 0.13-Cu/AG scaffold. Q_loss is thermal energy lost to the surroundings:

$$Q_{loss}=hA\Delta T$$ (Equation 3)

where h is the heat transfer coefficient, A is the surface area of the container, and ΔT is the temperature change, which is defined as T-T_surr (T and T_surr are the solution temperature and ambient temperature of the surroundings, respectively). Q_s is the heat associated with the light absorbed by solvent. At the maximum steady-state temperature, the heat input is equal to the heat output, so the equation can be:

$$Q_{\text{Cu}/\text{AG}}+Qs=Q_{loss}=hA\Delta T_{\max }$$ (Equation 4)

where ΔT_max is the temperature change at the highest steady-state temperature. According to the Equations 2 and 4, η can be determined:

$$\eta =\frac{hA\Delta T_{\max }-Qs}{I\left(1-{10}^{-A\lambda }\right)}$$ (Equation 5)

In this equation, only hA is unknown for calculation. In order to get hA, we herein introduce θ, which is defined as the ratio of ΔT to ΔT_max:

$$\theta =\frac{\Delta T}{\Delta T_{\max }}$$ (Equation 6)

Substituting Equation 6 into Equation 1, and rearranging Equation 1:

$$\frac{d\theta }{dt}=\frac{hA}{\sum _i{m}_i{C}_{p,i}}\left[\frac{Q_{\text{Cu}/\text{AG}}+Qs}{hA\Delta T_{\max }}-\theta \right]$$ (Equation 7)

When the laser was turned off, the Q_Cu/AG + Q_s = 0, so Equation 7 changed to:

$$dt=-\frac{\sum _i{m}_i{C}_{p,i}}{hA}\frac{d\theta }{\theta }$$ (Equation 8)

Integrating Equation 8 gives the expression:

$$t=-\frac{\sum _i{m}_i{C}_{p,i}}{hA}\theta$$ (Equation 9)

Thus, hA can be determined by applying the linear time data from the cooling period vs. –lnθ (Figure S7). By substituting the hA value into Equation 5, the photothermal conversion efficiency (η) of the 0.13-Cu/AG scaffold can be calculated.

In vitro degradation and copper ion release

For in vitro degradation testing, incubate the scaffolds in PBS solution at 37°C. The ratio of PBS volume to scaffold mass was 20 mL/g, and PBS was refreshed daily. Among them, the 0.13-Cu/AG scaffold was selected for degradation testing in PBS solutions with different pH values (6.5, 7.0, and 7.4). Then calculate the relative weight of the scaffolds. The remaining solution was collected to measure copper ion release using an Avio 200 ICP-OES System (PerkinElmer Inc., USA).

Organoid culture

Thyroid cancer organoids were generated following procedures previously reported by our group.^33,34 Briefly, collagenase digestion was used to isolate cells and small cell clusters from thyroid cancer samples. Mechanical digestion with a plastic pasteur pipette (320411, SORFA Life Science, China) was performed every 10 min during incubation. The mixture was then filtered using a 70-μm nylon mesh (258368, NEST Biotechnology, China), and embedded in Matrigel (356231, Corning). Thyroid cancer organoids were cultured in a basic growth medium consisting adDMEM/F12⁺⁺⁺ medium, 1 × B27 (17504-044, Gibco), 500 ng/mL of R-spondin-1 (120-38, Peprotech), 50 ng/mL of EGF (AF-100-15, Peprotech), 100 ng/mL of Noggin (120-10C, Peprotech), and 10 μM of SB202190 (S7067, Sigma-Aldrich) as the main components. Additionally, 10 μM of Y-27632 dihydrochloride (M1817, Abmole, USA) was added to the growth medium during the first 2 weeks of culture. The medium was refreshed every 3 days, and organoids were passaged at a ratio of 1:2-1:3 about every 2 weeks.

Histology

Primary tumor tissues and the derived organoids were fixed in 10% formalin at 4°C for 24 h, followed by dehydration, paraffin-embedding, and serial sectioning at a thickness of 4-μm. Histological analysis of all the tumor and organoid samples was performed by a board-certified pathologist. Histopathological diagnosis was made according to the standard classification system.

Immunofluorescence staining

Tissue and organoid slides were subjected to immunofluorescence analysis using standard procedures. The sections were incubated with primary antibodies against cytokeratin 19 (CK19; 1:200 dilution, AF5106, Affinity Biosciences), galectin-3 (1:300 dilution, ab76245, Abcam), and Ki-67 (1:300 dilution, ab16667, Abcam) at 4°C overnight. The sections were subsequently incubated with the secondary antibody Cy3-labeled goat anti-rabbit IgG (H + L) (1:300 dilution, A0516, Beyotime, China) for 1 h in the dark at room temperature. Nuclei were counterstained with DAPI for 10 min. Images were captured with a Carl Zeiss immunofluorescent microscope.

Treatment of cell lines and organoids with fiber scaffolds

Thyroid cancer cells were seeded into 24-well plates at a density of 1 × 10⁴ cells per well and cultured at for 24 h. The scaffolds loaded with different concentrations of copper ions were coculture with cells for 24 h. Scaffolds were further exposed to NIR laser irradiation at 1.3 W/cm² for 2.5 min. After incubation for another 12 h, the cell viability was measured by Cell Counting Kit-8 (CCK-8; HY-K0301, MedChemExpress, USA) cell cytotoxicity assay. According to the experimental results, scaffold with 0.13 mol/L copper ions was selected for the next experiment.

The scaffolds were added to cell culture plates and divided into six treatment groups: (1) Cu²⁺-loaded hydrogel scaffold with irradiation (Cu/AG + NIR); (2) Cu²⁺-loaded hydrogel scaffold without irradiation (Cu/AG); (3) hydrogel scaffold with irradiation (AG + NIR); (4) hydrogel scaffold without irradiation (AG); (5) without any treatment (Control).

Thyroid cancer organoids were resuspended in 75% matrigel/growth medium (1,000 organoids per drop) and plated in triplicate in 24-well plates. On the third day, the scaffold loaded with 0.13 mol/L copper ions was cocultured with organoids. The treatment method is the same as described above for the cell experiments. The viability of organoids was assessed using CellTiter-Glo 3D Reagent (G9683, Promega) in accordance with the manufacturer’s instructions, and luminescence readings were measured using a microplate reader (BioTek, USA).

For live and dead cell staining, cells or organoids were double-stained in the dark with 5 μM of calcein-AM (425201, Biolegend, USA) and 5 μM of propidium iodide (PI) (421301, Biolegend) for 20 min. Then, the cells or organoids were washed twice with PBS, and observed with a Carl Zeiss immunofluorescent microscope.

The level of intracellular ROS in BCPAP and CAL-62 cells was measured with a ROS Assay Kit (S0033S, Beyotime, China) according to the manufacture' instructions. 2’,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) was used to detect the ROS generation effect of the Cu/AG scaffolds. The fluorescence intensity in each well was detected by a fluorescence microplate reader and expressed as arbitrary units.

Quantification and statistical analysis

All data were obtained from at least three independent experiments, and results were expressed as mean ± standard deviation (SD) using one-way ANOVA analysis. Differences were considered statistically significant at ∗p < 0.05 and ∗∗p < 0.01.

Published: September 27, 2023

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.