Progettare microfabbriche di ossigeno per rallentare la progressione del tumore attraverso microambienti iperossici

Nature Communications volume 13, numero articolo: 4495 (2022) Citare questo articolo

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Mentre l’ipossia promuove la carcinogenesi, l’aggressività del tumore, la metastasi e la resistenza ai trattamenti oncologici, gli impatti dell’iperossia sui tumori sono raramente esplorati perché fornire un apporto di ossigeno a lungo termine in vivo è una sfida importante. Qui, costruiamo micro fabbriche di ossigeno, vale a dire microcapsule di fotosintesi (PMC), mediante incapsulamento di cianobatteri acquisiti e nanoparticelle di upconversion in microcapsule di alginato. Questo sistema consente un apporto di ossigeno duraturo attraverso la conversione della radiazione esterna in emissioni di lunghezza d’onda rossa per la fotosintesi nei cianobatteri. Il trattamento con PMC sopprime la via NF-kB, la produzione di HIF-1α e la proliferazione delle cellule tumorali. Il microambiente iperossico creato da un impianto PMC in vivo inibisce la crescita e le metastasi dell’epatocarcinoma e ha effetti sinergici insieme all’anti-PD-1 nel cancro al seno. Le fabbriche di ossigeno ingegneristico offrono potenziale per studi sulla biologia dei tumori in microambienti iperossici e ispirano l’esplorazione di trattamenti oncologici.

L’ipossia è la caratteristica più pervasiva dei microambienti dei tumori solidi1,2 e deriva da uno squilibrio tra un apporto insufficiente di ossigeno e un aumento del consumo di ossigeno da parte delle cellule tumorali in rapida proliferazione. Di conseguenza, le cellule tumorali ricorrono a molteplici percorsi adattativi e cambiamenti genomici per sopravvivere in ambienti ipossici3. Il fattore di trascrizione fattore 1α inducibile dall'ipossia (HIF-1α), il mediatore più riconosciuto delle risposte ipossiche, svolge un ruolo centrale nello stimolare la neovascolarizzazione nei tumori per migliorare l'apporto di ossigeno e nutrienti4. Paradossalmente, questi vasi sono spesso organizzati in modo irregolare (ad esempio, strutture contorte, iperpermeabili e con estremità cieca) e presentano difetti nella diffusione o nella perfusione dell'ossigeno5, con conseguente espansione delle regioni ipossiche nei tumori. Allo stesso tempo, è stato segnalato che il microambiente ipossico, un segno distintivo dei tumori maligni, è non solo la barriera primaria che protegge il tumore da varie terapie creando un ambiente di immunosoppressione6, attivando il percorso di riparazione del DNA7 e consentendo il flusso autofagico8 ma anche un promotore della carcinogenesi9 , invasività del tumore e metastasi1,2. Questi risultati hanno ispirato l’esplorazione di tecnologie per convertire microambienti ipossici in microambienti iperossici per studi sulla biologia o sulla terapia del tumore.

Costruire un microambiente iperossico di lunga durata nei tumori è una sfida importante a causa della mancanza di fonti di ossigeno costanti e biocompatibili. Considerando che i microbi algali sono i principali fornitori di O2 sulla Terra, la fotosintesi nei cloroplasti algali potrebbe essere potenzialmente esplorata per integrare O2 nei tumori. Il macchinario fotosintetico richiede una sorgente luminosa adatta che emette fotoni da 650–700 nm. Poiché le nanoparticelle di upconversion (UCNP) basate sulle terre rare hanno mostrato una straordinaria capacità di convertire i laser biotrasparenti del vicino infrarosso (NIR) in luce visibile10, questi materiali potrebbero essere sfruttati per fornire fotoni disponibili nella fotosintesi. Abbiamo quindi ipotizzato che un microambiente iperossico di lunga durata potrebbe essere creato mediante la costruzione razionale di microbi algali e UCNP.

In questo studio, siamo pionieri di una microcapsula di fotosintesi (PMC) incapsulando cianobatteri e UCNP in microcapsule di alginato (MC) che possono essere fabbricate mediante una tecnica di goccioline elettrostatiche. Quattro ceppi di cianobatteri sono stati soggetti a selezione di acclimatazione per acquisire un ceppo adatto all'adattamento alle condizioni fisiologiche. Esploriamo in modo completo gli impatti delle radiazioni NIR, della popolazione cellulare e della dose di UCNP sulla produzione di O2 per progettare una formula ottimizzata di PMC. Gli impatti dei microambienti iperossici creati dalle PMC vengono esaminati in nove linee cellulari tumorali e due modelli tumorali, tra cui il cancro al seno ortotopico nei topi e l'epatocarcinoma trapiantato nei conigli.

32 °C, S. sp. 6803 and S. elongate. 7942 were able to acclimate to the temperature increments. Stepwise changes from the BG11 medium to DMEM allowed us to acquire an evolved S. sp. 6803 (e-S. sp. 6803) strain, which maintained its activity at 37 °C in DMEM (Supplementary Fig. 3). This strain was therefore selected for PMC construction. We then synthesized a series of UCNPs with different emissions by the crystal growth method10. An Er3+- and Yb3+-doped NaYF4 nanorod (15.3 × 30.2 nm) was found to emit strong fluorescence at 660 nm, perfectly matching the absorbance of chlorophyll α (Supplementary Fig. 4 and Supplementary Fig. 5), which is the prominent component responsible for photosynthesis. Next, we engineered the PMCs by encapsulating algal microbes and UCNPs in the alginate-calcium microspheres under an electrostatic field via an electrostatic droplet generation system. As shown in Fig. 1, the whole process involves four steps: (i) homogenous mixing of algal microbes with UCNPs in alginate sodium; (ii) dispersing of alginate sodium solution into uniform droplets under an electrostatic field; (iii) encapsulating UCNPs and microbes by cross-linked alginate-calcium in CaCl2 solutions; and (iv) coating of alginate-calcium microspheres by poly-L-lysine (PLL), a water-soluble polycation that is resistant to enzymatic degradation and capable of preventing microbe leakage (Supplementary Fig. 6). The resulting PMCs were examined by upconversion luminescence microscopy. While empty MCs and alga-encapsulated MCs had no fluorescence signals, PMCs emitted strong red fluorescence under 980 nm excitation (Fig. 2a). These results indicated that the encapsulated UCNPs thoroughly maintained their optical properties. We comprehensively examined the impacts of microbe density and UCNP concentration on the photosynthetic activity of PMCs (Fig. 2b). An optimized formula of PMCs (3 × 103 algal cells and 0.67 μg of UCNPs per MC) was acquired for efficient oxygen production (1.6 μg/min). The oxygen generation of designated PMCs was dependent on the intensity and exposure time of NIR radiation, indicating a controllable oxygen supply (Supplementary Fig. 7). The encapsulated algal microbes in PMCs survived in DMEM for over 1 month (Supplementary Fig. 8)./p>140 days and were almost cured, as there were no detectable tumour nodules in the livers and lungs by CT imaging and ex vivo examination (Supplementary Fig. 23). To our surprise, luminescent PMCs could still be observed in the liver and maintained spherical morphologies (Supplementary Fig. 23). These results indicated that the hyperoxic microenvironment created by NIR-PMCs could greatly slow tumour progression, inhibit tumour metastasis and enhance the survival rates of hepatocarcinoma-bearing rabbits./p>140 days) than the untreated animals did (average survival time ~27 days) and had no detectable tumour nodes. However, these findings may need more validation across different tumour models./p>Stage II) to establish local control and palliation. The PMCs were deliberately designed to accommodate the interventional device. Although intratumor injection was selected for the administration of PMCs in animals, PMCs could be applied in human patients by transcatheter arterial chemoembolization. To facilitate future clinical applications, the dose and duration time of NIR radiation should be carefully examined. Taking hepatocarcinoma as an example, 900 mW/cm2 NIR radiation at 980 nm was applied to rabbits for 60 min/day. Given that the depth of hepatocarcinoma in rabbits is 0.3–0.7 cm, the tumour received 300–500 mW/cm2 radiation after penetration of the animal belly60. To acquire such excitation intensity in human patients, the radiation dose has to be increased to 2000 mW/cm2 or the duration time should be extended to 180 min because hepatocarcinoma in human patients are often deeper than they are in rabbits61,62,63 and because the intensity of NIR radiation at 980 nm would decline to <30% after penetration of 0.7–1.0 cm belly tissues in humans. However, this amount of NIR radiation may induce hyperthermia damage. Alternatively, NIR-II radiation at 1200–1700 nm would be more suitable for clinical applications, as NIR-II photons have a much deeper penetration capability than NIR lasers at 980 nm64. UCNPs with excitations in the NIR-II region could be exploited to construct PMCs for potential applications in clinics. Since interventional therapy is a conventional treatment in hepatocarcinoma patients, PMCs are a promising implant for clinical applications./p>85% cell proliferation, we increased the culture temperature (1 °C). Otherwise, we sustained the temperature for another 24 h. After 30 days culture, the evolved algal microbes were acquired and preserved in BG11 media at 37 °C for subsequent tests. Next, we repeated the above procedure by stepwise changing medium composition from BG11 to DMEM. The evolved Synechocystis sp. 6803 cultured at 37 °C in DMEM was denoted as e-S. sp. 6803./p>50 μm./p>2 cm for mice or the tumour volume is >80 cm3 for rabbits; (ii) the eating, drinking or movement of animals is severely affected. To develop the hepatic VX2 tumours in rabbits, VX2 cell suspensions (2 × 106 cells, 200 μL) were implanted into the thigh muscles of donor rabbits. Once the tumour sizes were >2 cm (~2 weeks), the donor rabbits were anesthetized by intravenous injection at a lethal dose 2 mL/Kg of xylazine hydrochloride for the harvest of tumour tissues. Each tumour was minced into 1 mm3 piece by ophthalmic scissors under sterile conditions. The recipient rabbits were anesthetized by intramuscular injection of xylazine hydrochloride (250 μL/Kg). A minced tissue fragment was directly delivered percutaneously into the subcapsular parenchyma of the left hepatic lobe of the recipient rabbit by percutaneous puncture technique under a 16-slice CT spiral scan (Brilliance-16, Phillips, USA) guidance. The rabbits were housed and examined by CT imaging until the tumour volumes reached around 1 cm3. The hepatocarcinoma-bearing rabbits with similar tumour size were divided into two groups by throwing dice, including vehicle control (n = 13), NIR-PMC group (n = 13). The rabbits were anesthetized for a single intratumorally injection of PMC suspensions (500 µL, 3.6 × 104/mL) at 14 days. NIR radiations at 900 mW/cm2 were exposed to animals for three intervals (20 min in each interval) each day. The tumour size was monitored by CT scanning (MHCT brilliance 16, Philips, Holland) every 2 weeks./p> 1 indicates antagonism, CI = 1 indicates additivity, and CI < 1 indicates synergy. The tumours were imaged by IVIS imaging spectrum system (PerkinElmer, ME, USA) and Canon camera (Japan). The mice were fully anesthetized by an overdose of sodium pentobarbital (400 mg/kg) and sacrificed to collect tumours and lungs. The tissue samples were stored in liquid nitrogen for cytokine and adenosine measurements or fixed for H&E staining or immunostaining of A2AR, CD4, CD39, CD206 and CD73 expression./p>