Impacting Pancreatic Cancer Therapy in Heterotypic in Vitro Organoids and in Vivo Tumors with Specificity-Tuned, NIR-Activable Photoimmunonanoconjugates: Towards Conquering Desmoplasia?

Abstract

Despite untiring efforts to develop therapies for pancreatic ductal adenocarcinoma (PDAC), survival statistics remain dismal, necessitating distinct approaches. Photodynamic priming (PDP), which improves drug delivery and combination regimens, as well as tumor photodestruction are key attributes of photodynamic therapy (PDT), making it a distinctive clinical option for PDAC. Localized, high-payload nanomedicine-assisted delivery of photosensitizers (PSs), with molecular specificity and controlled photoactivation, thus becomes critical in order to reduce collateral toxicity during more expansive photodynamic activation procedures with curative intent. As such, targeted photoactivable lipid-based nanomedicines are an ideal candidate but have failed to provide greater than twofold cancer cell selectivity, if at all, due to their extensive multivariant physical, optical, and chemical complexity. Here, we report (1) a systematic multivariant tuning approach to engineer (Cet, anti-EGFR mAb) photoimmunonanoconjugates (PINs), and (2) stroma-rich heterotypic PDAC in vitro and in vivo models incorporating patient-derived pancreatic cancer-associated fibroblasts (PCAFs) that recapitulate the desmoplasia observed in the clinic. These offer a comprehensive, disease-specific framework for the development of Cet-PINs. Specificity-tuning of the PINs, in terms of PS lipid anchoring, electrostatic modulation, Cet orientation, and Cet surface densities, achieved ∼16-fold binding specificities and rapid penetration of the heterotypic organoids within 1 h, thereby providing a ∼16-fold enhancement in molecular targeted NIR photodestruction. As a demonstration of their inherent amenability for multifunctionality, encapsulation of high payloads of gemcitabine hydrochloride, 5-fluorouracil, and oxaliplatin within the Cet-PINs further improved their antitumor efficacy in the heterotypic organoids. In heterotypic desmoplastic tumors, the Cet-PINs efficiently penetrated up to 470 μm away from blood vessels, and photodynamic activation resulted in substantial tumor necrosis, which was not elicited in T47D tumors (low EGFR) or when using untargeted constructs in both tumor types. Photodynamic activation of the Cet-PINs in the heterotypic desmoplastic tumors resulted in collagen photomodulation, with a 1.5-fold reduction in collagen density, suggesting that PDP may also hold potential for conquering desmoplasia. The in vivo safety profile of photodynamic activation of the Cet-PINs was also substantially improved, as compared to the untargeted constructs. While treatment using the Cet-PINs did not cause any detriment to the mice's health or to healthy proximal tissue, photodynamic activation of untargeted constructs induced severe acute cachexia and weight loss in all treated mice, with substantial peripheral skin necrosis, muscle necrosis, and bowel perforation. This study is the first report demonstrating the true value of molecular targeting for NIR-activable PINs. These constructs integrate high payload delivery, efficient photodestruction,...

An Organotypic Microcosm for the Pancreatic Tumor Microenvironment

“…A useful method for studying tumor-stroma interactions includes cell line or human tissue-derived organoids cocultured with commercial fibroblasts lines or fibroblasts isolated from patients [4,12,[23][24][25][26]. Several publications have reported using cell line-derived organoids cultured with tumor-associated fibroblasts to study drug delivery systems and treatment resistance in pancreatic cancer [4,[23][24][25]. Cell line-derived organoids cultured with fibroblasts had heterogenous distribution in size and shape.…”

“…Moreover, coculture studies have been used to test novel photodynamic drug delivery systems [23,33]. One report utilizes heterotypic PDAC organoids created from MIAPaCa-2 cells cultured with patient-derived pancreatic CAFs as a model for testing a novel cetuximab photoimmunonanoconjugate delivery system [23].…”

“…Moreover, coculture studies have been used to test novel photodynamic drug delivery systems [23,33]. One report utilizes heterotypic PDAC organoids created from MIAPaCa-2 cells cultured with patient-derived pancreatic CAFs as a model for testing a novel cetuximab photoimmunonanoconjugate delivery system [23]. Cetuximab is a monoclonal antibody that targets EGFR, which is overexpressed in many PDAC tumors and CAFs, and therefore may serve as a valuable target to eliminate the desmoplastic barrier [23,[34][35][36].…”

“…One report utilizes heterotypic PDAC organoids created from MIAPaCa-2 cells cultured with patient-derived pancreatic CAFs as a model for testing a novel cetuximab photoimmunonanoconjugate delivery system [23]. Cetuximab is a monoclonal antibody that targets EGFR, which is overexpressed in many PDAC tumors and CAFs, and therefore may serve as a valuable target to eliminate the desmoplastic barrier [23,[34][35][36]. The cetuximab photoimmunonanoconjugate efficiently penetrated PDAC organoid-fibroblast cultures after 1 hour of incubation [23].…”

“…Cetuximab is a monoclonal antibody that targets EGFR, which is overexpressed in many PDAC tumors and CAFs, and therefore may serve as a valuable target to eliminate the desmoplastic barrier [23,[34][35][36]. The cetuximab photoimmunonanoconjugate efficiently penetrated PDAC organoid-fibroblast cultures after 1 hour of incubation [23]. Further, by inhibiting EGFR, which is necessary and sufficient for desmoplastic reaction [21], with cetuximab, this technique may provide an avenue of tumor penetration and destruction when coupled with chemotherapy.…”