Thyroid cancer (TC) is the most common endocrine tumor, with a rapidly increasing incidence in recent years. Anaplastic thyroid cancer (ATC) accounts for approximately 1–2 % of all TC, but the prognosis of ATC is extremely poor, with a median survival of 5 months and a 1 year survival rate of only 20 % [1,2]. In general, ATC requires a multidisciplinary therapy including surgery, chemotherapy, and radiotherapy. However, at the time of diagnosis, about 85–95 % of ATC primary tumors have cervical lymph node metastases and extensive invasion of cervical structures, which increases the difficulty of surgery, affects the quality of life, and even deprives patients of the opportunity to undergo surgery [1,3,4]. For such ATCs, systemic therapies such as chemotherapy or molecular targeted treatments are chosen, but the response rate is very low, which is difficult to effectively extend patients' overall survival [5]. Immunotherapy has brought new hope for the treatment of ATC [6]. Immune checkpoint blockade (ICB) targeting programmed cell death protein 1 (PD1) and programmed cell death-ligand 1 (PD-L1) has been applied for the treatment of ATC. However, clinical study results show that only 10–15 % of patients respond to PD1 blockade therapy [7]. In some patients, the response rate is low, making it difficult for ATC patients to benefit from long-term treatment [7]. Therefore, it is worth to explore or develop combined immunotherapy for the improved therapeutic efficacy for ATC.

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME), and their infiltration is significantly correlated with tumor progression, poor prognosis, and other adverse clinical outcomes [8,9]. TAMs in the TME can be broadly divided into two phenotypes: one type is M1 that exerts anti-tumor effects and the other type is M2 that promotes tumor growth. M2-like TAMs are the most represented phenotype in TAMs, which mediate tumor immune evasion and angiogenesis, enhancing the invasiveness of tumor cells and promoting tumor growth and metastasis [10,11]. Besides driving tumor progression, M2-like TAMs secrete cytokines to recruit immunosuppressive cells (e.g., Tregs, Th2) and block CD8+ T cell infiltration, further dampening anti-tumor immunity [8,12,13].

Phosphatidylinositol 3-kinase gamma (PI3Kγ) is highly expressed in myeloid cells, and can activate CCAAT enhancer binding protein β (CLEBPβ) to induce tumor immune suppression transcriptional programs, and inhibit the pro-inflammatory effects of nuclear factor kappa-B (NF-κB) proteins, thereby suppressing anti-tumor immunity [14,15]. Blockade of PI3Kγ reduces the accumulation of M2-like TAMs by decreasing their chemotaxis, inhibiting their immunosuppressive function, and altering the immune status of the TME [14]. Eganelisib (IPI549) is a highly selective PI3Kγ inhibitor, which can enhance anti-tumor immune responses, thereby inhibiting tumor growth and metastasis [16]. In addition, IPI549 can synergize with the anti-tumor effects of anti-PD1/anti-PD-L1 immunotherapy and overcome tumor resistance [16,17]. Thus, IPI549 not only has the potential to act as a monotherapeutic agent for tumors, but also as an immune adjuvant to promote the efficacy of immunotherapy. However, the results of available clinical studies show that IPI549 shows modest single-agent activity only when its concentration exceeds its IC90 value [18]. In addition, treatment-related serious adverse events such as liver injury, fever and rash were observed in about 18 % of patients using IPI549 [18]. The above results suggest that the antitumor effect of IPI549 is limited and dose-dependent with biotoxicity. Therefore, achieving targeted delivery of IPI549 and improving its tumor delivery efficiency are key aspects to improve the safety and utilization of IPI549.

Ultrasound-targeted microbubble destruction (UTMD) mediated drug delivery is a safe and highly cost-effective treatment method [19,20]. After intravenous injection of drug-loaded microbubbles (MBs), UTMD is applied at the tumor site, mediating stable cavitation or inertial cavitation and promoting the delivery and penetration of drugs into the tumor through various biological effects [19]. Tumor-targeted peptides and antibodies are applied to modify the surface of MBs to construct targeted microbubbles (tMBs), which can specifically target tumor tissues. Combined with UTMD, this achieves dual-targeted delivery of drugs. Integrins αvβ3 are expressed in angiogenic endothelial cells and most tumor cells, not in normal tissue resting endothelial cells, and can be used as targets for tumor-specific imaging and therapy [21]. On the one hand, targeting integrins αvβ3 can increase the accumulation of drug-carrying MBs in the tumor before UTMD and improve drug release at the tumor; on the other hand, phospholipid fragments or nanoparticles carrying the drug can be re-targeted to the tumor along with the blood circulation after UTMD, which maximizes drug utilization and reduces its impact on normal tissues.

In general, given the current therapeutic dilemma and pathological characteristics of ATC, this study aims to construct integrin αvβ3 targeted microbubbles loaded with IPI549 and ICG, IPI549@αIMBs, combined with UTMD, for the theranostics of ATC (Fig. 1). Relying on active targeting of integrins αvβ3, it serves as a delivery vehicle of IPI549 to the tumor vessels of ATC. Subsequently, UTMD is further combined to achieve the controlled release of IPI549 specifically at the ATC tumor site, realizing the dual-targeted delivery of IPI549 and monitored through contrast enhanced ultrasound. On the other hand, IPI549@αIMBs, as a contrast agent for contrast enhanced ultrasound (CEUS), can visualize the targeted release of IPI549 at the ATC site; by doping the membrane component with the ICG, the systemic distribution of the drug can be monitored through fluorescence molecular imaging (FMI) after UTMD, to assess the treatment effectiveness and safety of IPI549 delivery in real-time. This study aims to verify the feasibility of our hypotheses and to explore new therapeutic methods for ATC.

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)2000-N-hydroxysuccinimide ester (DSPE-PEG2000-NHS), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine conjugated with Cyanine5.5 (DSPE-Cy5.5), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine conjugated with Indocyanine Green (DSPE-ICG), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)2000 (DSPE-PEG2000) were obtained from Ruixi Biotechnology Co., Ltd. (Xi'an, China). Cyclo (Arg-Gly-Asp-Tyr-Lys) (c(RGD)yk) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). IPI549 was obtained from Selleck Chemicals (Catalog No. S8330). In vivo anti-mouse PD1 antibody was obtained from BioXell (CD279, Catalog No. BE0146).

Human thyroid normal cells Nthy-ori 3–1 and human metaplastic thyroid cancer cells Cal-62 were supplied by BNCC company (Beijing, China). C57BL/6-derived mouse ATC cell line BPC (FD-MT-BPC, Fudan Cancer-Mouse Thyroid-BRAF/P53 Cre) was kindly provided by Professor Yu-Long Wang from Fudan University Shanghai Cancer Center [6,22]. BALB/c nude mice and C57BL/6 mice were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. Each animal study was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee at the China-Japan Friendship Hospital (No. zryhyy21–23–02-01).