Photodynamic Priming Research Articles

PpIX-enabled fluorescence-based detection and photodynamic priming of platinum-resistant ovarian cancer cells under fluid shear stress.

Over 75% percent of ovarian cancer patients are diagnosed with advanced-stage disease characterized by unresectable intraperitoneal dissemination and the presence of ascites, or excessive fluid build-up within the abdomen. Conventional treatments include cytoreductive surgery followed by multi-line platinum and taxane chemotherapy regimens. Despite an initial response to treatment, over 75% of patients with advanced-stage ovarian cancer will relapse and succumb to platinum-resistant disease. Recent evidence suggests that fluid shear stress (FSS), which results from the movement of fluid such as ascites, induces epithelial-to-mesenchymal transition and confers resistance to carboplatin in ovarian cancer cells. This study demonstrates, for the first time, that FSS-induced platinum resistance correlates with increased cellular protoporphyrin IX (PpIX), the penultimate downstream product of heme biosynthesis, the production of which can be enhanced using the clinically approved pro-drug aminolevulinic acid (ALA). These data suggest that, with further investigation, PpIX could serve as a fluorescence-based biomarker of FSS-induced platinum resistance. Additionally, this study investigates the efficacy of PpIX-enabled photodynamic therapy (PDT) and the secretion of extracellular vesicles under static and FSS conditions in Caov-3 and NIH:OVCAR-3 cells, two representative cell lines for high-grade serous ovarian carcinoma (HGSOC), the most lethal form of the disease. FSS induces resistance to ALA-PpIX-mediated PDT, along with a significant increase in the number of EVs. Finally, the ability of PpIX-mediated photodynamic priming (PDP) to enhance carboplatin efficacy under FSS conditions is quantified. These preliminary findings in monolayer cultures necessitate additional studies to determine the feasibility of PpIX as a fluorescence-based indicator, and mediator of PDP, to target chemoresistance in the context of FSS.

Overcoming the effects of fluid shear stress in ovarian cancer cell lines: Doxorubicin alone or photodynamic priming to target platinum resistance.

Resistance to platinum-based chemotherapies remains a significant challenge in advanced-stage high-grade serous ovarian carcinoma, and patients with malignant ascites face the poorest outcomes. It is, therefore, important to understand the effects of ascites, including the associated fluid shear stress (FSS), on phenotypic changes and therapy response, specifically FSS-induced chemotherapy resistance and the underlying mechanisms in ovarian cancer. This study investigated the effects of FSS on response to cisplatin, a platinum-based chemotherapy, and doxorubicin, an anthracycline, both of which are commonly used to manage advanced-stage ovarian cancer. Consistent with prior research, OVCAR-3 and Caov-3 cells cultivated under FSS demonstrated significant resistance to cisplatin. Examination of the role of mitochondria revealed an increase in mitochondrial DNA copy number and intracellular ATP content in cultures grown under FSS, suggesting that changes in mitochondria number and metabolic activity may contribute to platinum resistance. Interestingly, no resistance to doxorubicin was observed under FSS, the first such observation of a lack of resistance under these conditions. Finally, this study demonstrated the potential of photodynamic priming using benzoporphyrin derivative, a clinically approved photosensitizer that localizes in part to mitochondria and endoplasmic reticula, to enhance the efficacy of cisplatin, but not doxorubicin, thereby overcoming FSS-induced platinum resistance.

Photodynamic priming modulates cellular ATP levels to overcome P-glycoprotein-mediated drug efflux in chemoresistant triple-negative breast cancer.

P-glycoprotein (P-gp, ABCB1) is a well-researched ATP-binding cassette (ABC) drug efflux transporter linked to the development of cancer multidrug resistance (MDR). Despite extensive studies, approved therapies to safely inhibit P-gp in clinical settings are lacking, necessitating innovative strategies beyond conventional inhibitors or antibodies to reverse MDR. Photodynamic therapy is a globally approved cancer treatment that uses targeted, harmless red light to activate non-toxic photosensitizers, confining its cytotoxic photochemical effects to disease sites while sparing healthy tissues. This study demonstrates that photodynamic priming (PDP), a sub-cytotoxic photodynamic therapy process, can inhibit P-gp function by modulating cellular respiration and ATP levels in light accessible regions. Using chemoresistant (VBL-MDA-MB-231) and chemosensitive (MDA-MB-231) triple-negative breast cancer cell lines, we showed that PDP decreases mitochondrial membrane potential by 54.4% ± 30.4 and reduces mitochondrial ATP production rates by 94.9% ± 3.46. Flow cytometry studies showed PDP can effectively improve the retention of P-gp substrates (calcein) by up to 228.4% ± 156.3 in chemoresistant VBL-MDA-MB-231 cells, but not in chemosensitive MDA-MB-231 cells. Further analysis revealed that PDP did not alter the cell surface expression level of P-gp in VBL-MDA-MB-231 cells. These findings indicate that PDP can reduce cellular ATP below the levels that is required for the function of P-gp and improve intracellular substrate retention. We propose that PDP in combination with chemotherapy drugs, might improve the efficacy of chemotherapy and overcome cancer MDR.

Minocycline and photodynamic priming significantly improve chemotherapy efficacy in heterotypic spheroids of pancreatic ductal adenocarcinoma

The prognosis for patients with advanced-stage pancreatic ductal adenocarcinoma (PDAC) remains dismal. It is generally accepted that combination cancer therapies offer the most promise, such as Folforinox, despite their associated high toxicity. This study addresses the issue of chemoresistance by introducing a complementary dual priming approach to attenuate the DNA repair mechanism and to improve the efficacy of a type 1 topoisomerase (Top1) inhibitor. The result is a regimen that integrates drug-repurposing and nanotechnology using 3 clinically relevant FDA-approved agents (1) Top1 inhibitor (irinotecan) at subcytotoxic doses (2) benzoporphyrin derivative (BPD) as a photoactive molecule for photodynamic priming (PDP) to improve the delivery of irinotecan within the cancer cell and (3) minocycline priming (MNP) to modulate DNA repair enzyme Tdp1 (tyrosyl-DNA phosphodiesterase) activity. We demonstrate in heterotypic 3D cancer models that incorporate cancer cells and pancreatic cancer-associated fibroblasts that simultaneous targeting of Tdp1 and Top1 were significantly more effective by employing MNP and photoactivatable multi-inhibitor liposomes encapsulating BPD and irinotecan compared to monotherapies or a cocktail of dual or triple-agents. These data are encouraging and warrant further work in appropriate animal models to evolve improved therapeutic regimens.

Remediating Desmoplasia using Targeted Photo-nanomedicines: Improving Surgical and Survival Outcomes

SignificanceIn recent years, a deeper understanding of the sub-therapeutic photodynamic priming (PDP) effects of photodynamic therapy is being gained. Here we show that PDP can remediate desmoplasia in pancreatic and head and neck tumors, improving both surgical and survival outcomes. ApproachOrthotopic FaDu head and neck tumors and MIA-PaCa-2 pancreatic tumors were used. PDP is facilitated by various nanoconstructs containing benzoporphyrin derivative and its lipidated variants, in addition to molecular targeting provided by conjugates of Cetuximab (anti-EGFR monoclonal antibody). ResultsPDP improved the tumor delivery and diagnostic accuracy of the clinical fluorescence image-guided surgery probe Cetuximab-IRDye800CW by 264.2%. Time-to-surgery is reduced from 25.7 h to 2.5 h. PDP also reduced the density (>90%) and alignment (>103-fold) of collagen in pancreatic tumors. PDP induced 90% tumor growth inhibition at 8.1% of the clinical chemotherapy dose and doubled overall survival. ConclusionsThis study demonstrates how remediating desmoplasia using PDP plays a versatile role in cancer management, from improving image guided surgery to potentiating chemotherapy.

Using photodynamic approaches to improve the diagnostic accuracy of image guided surgery

SignificanceIncomplete surgical resection of tumors using white light and preoperative imaging causes tumor reoccurrence. Antibody-dye conjugate can target and accumulate into the tumor but has longer wait times for surgery following administration. This process can be expedited using PDP which not only increase the delivery but also improves the diagnostic accuracy in tumor detection. ApproachThe mice bearing FaDu head and neck tumors were administered with Cetuximab-IRDye800 (Cet-IRDye800) and treated with sub-therapeutic dose of PDT (photodynamic Priming; PDP). The accumulation and distribution of Cet-IRDye800 into the tumor were measured and analyzed. ResultsPDP improves and expedites the delivery of Cet-IRDye800 into the tumors significantly. It also improves diagnostic accuracy in tumor detection with respect to the vicinal salivary gland. ConclusionsThis study highlights, for the first time, that PDP enhances the rate and extend of delivery of the fluorescently labeled antibodies for the effective photodynamic image guided surgery.

Comparison of Photodynamic Priming Efficacy, with BPD or ALA-PpIX, to Overcome PFAS-Induced Platinum Resistance in Ovarian Cancer

Ovarian cancer is the most lethal gynecologic cancer, largely due to the development of platinum-based chemotherapy resistance. Understanding sources of chemoresistance and the development of mechanism-based treatments are critical. In a recent study, we reported that perfluoroalkyl substances (PFAS), endocrine disruptors that pollute drinking water supplies worldwide, induce carboplatin resistance in ovarian cancer cells, potentially by enhancing mitochondrial function. The ability of photodynamic priming (PDP) to overcome PFAS-induced resistance using photosensitizers that, in part, localize to mitochondria, specifically benzoporphyrin derivative (BPD) or aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX)), will be presented. Ovarian cancer cells were exposed to PFAS, then treated with either BPD- or ALA-PpIX-PDP followed by carboplatin. Under PFAS exposure conditions that previously induced chemoresistance, both BPD- and ALA-PpIX-PDP sensitized ovarian cancer cells to carboplatin. A concomitant decrease in mitochondrial membrane potential was observed.

Shedding Light on Chemoresistance: The Perspective of Photodynamic Therapy in Cancer Management.

The persistent failure of standard chemotherapy underscores the urgent need for innovative and targeted approaches in cancer treatment. Photodynamic therapy (PDT) has emerged as a promising photochemistry-based approach to address chemoresistance in cancer regimens. PDT not only induces cell death but also primes surviving cells, enhancing their susceptibility to subsequent therapies. This review explores the principles of PDT and discusses the concept of photodynamic priming (PDP), which augments the effectiveness of treatments like chemotherapy. Furthermore, the integration of nanotechnology for precise drug delivery at the right time and location and PDT optimization are examined. Ultimately, this study highlights the potential and limitations of PDT and PDP in cancer treatment paradigms, offering insights into future clinical applications.

PD-L1 Immune Checkpoint Targeted Photoactivable Liposomes (iTPALs) Prime the Stroma of Pancreatic Tumors and Promote Self-Delivery.

Desmoplasia in pancreatic ductal adenocarcinoma (PDAC) limits the penetration and efficacy of therapies. It has been previously shown that photodynamic priming (PDP) using EGFR targeted photoactivable multi-inhibitor liposomes remediates desmoplasia in PDAC and doubles overall survival. Here, bifunctional PD-L1 immune checkpoint targeted photoactivable liposomes (iTPALs) that mediate both PDP and PD-L1 blockade are presented. iTPALs also improve phototoxicity in PDAC cells and induce immunogenic cell death. PDP using iTPALs reduces collagen density, thereby promoting self-delivery by 5.4-fold in collagen hydrogels, and by 2.4-fold in syngeneic CT1BA5 murine PDAC tumors. PDP also reduces tumor fibroblast content by 39.4%. Importantly, iTPALs also block the PD-1/PD-L1 immune checkpoint more efficiently than free α-PD-L1 antibodies. Only a single sub-curative priming dose using iTPALs provides 54.1% tumor growth inhibition and prolongs overall survival in mice by 42.9%. Overall survival directly correlates with the extent of tumor iTPAL self-delivery following PDP (Pearson's r = 0.670, p = 0.034), while no relationship is found for sham non-specific IgG constructs activated with light. When applied over multiple cycles, as is typical for immune checkpoint therapy, PDP using iTPALs promises to offer durable tumor growth delay and significant survival benefit in PDAC patients, especially when used to promote self-delivery of integrated chemo-immunotherapy regimens.

A single photodynamic priming protocol augments delivery of ⍺-PD-L1 mAbs and induces immunogenic cell death in head and neck tumors.

Photodynamic priming (PDP) leverages the photobiological effects of subtherapeutic photodynamic therapy (PDT) regimens to modulate the tumor vasculature and stroma. PDP also sensitizes tumors to secondary therapies, such as immunotherapy by inducing a cascade of molecular events, including immunogenic cell death (ICD). We and others have shown that PDP improves the delivery of antibodies, among other theranostic agents. However, it is not known whether a single PDP protocol is capable of both inducing ICD in vivo and augmenting the delivery of immune checkpoint inhibitors. In this rapid communication, we show for the first time that a single PDP protocol using liposomal benzoporphyrin derivative (Lipo-BPD, 0.25 mg/kg) with 690 nm light (75 J/cm2 , 100 mW/cm2 ) simultaneously doubles the delivery of ⍺-PD-L1 antibodies in murine AT-84 head and neck tumors and induces ICD in vivo. ICD was observed as a 3-11 fold increase in tumor cell exposure of damage-associated molecular patterns (Calreticulin, HMGB1, and HSP70). These findings suggest that this single, highly translatable PDP protocol using clinically relevant Lipo-BPD holds potential for improving immunotherapy outcomes in head and neck cancer. It can do so by simultaneously overcoming physical barriers to the delivery of immune checkpoint inhibitors, and biochemical barriers that contribute to immunosuppression.

Abstract 3228: Photodynamic priming enhances immunotherapy responses overcoming oncogenic drivers in pancreatic patient derived tumor organoids

Abstract Photodynamic therapy (PDT) is an anti-cancer therapy that utilizes light, a photoresponsive photosensitizer (PS), and oxygen to confer direct cytotoxicity or vascular damage. PDT alters the tumor microenvironment (TME) transiently in a process termed photodynamic priming (PDP), making it more receptive to subsequent therapies. Previous studies reported that PDP exerts immune stimulatory effects via the release of damage associated molecular patterns (DAMPs) as a potent inducer of immunogenic cell death. Pancreatic cancer (PC) is a lethal human malignancy with no curable treatments available thus far. The desmoplastic stroma in PC limits the delivery of therapeutic agents contributing to treatment resistance. More, the stroma promotes an immunosuppressive TME that leads to immune exclusion, which is largely responsible for the immunotherapy failure in PC. Therefore, there is an unmet need to find more effective combination therapies against PC. In this work, we evaluated whether PDP enhances anti-PD1 therapy response by modulating immunogenicity in PC patient-derived organoids (PDOs). Visudyne (PS) and varied light doses of 25-100 J/cm2 were used for PDP treatments. Our data showed light dose dependent cytotoxicity and the expression of DAMPs including HSP60, calreticulin and HMGB1 in PDOs. Gene expression analysis of PDP treated PDOs showed increases in IFNγ, TNFα and CXCL12 while decreases in PDL1, TGFβ1 and FOXP1. Coculture of healthy donor (HD) monocyte-derived dendritic cells (mDCs) for 24 h with PDOs treated with a sublethal dose of PDP (25 J/cm2) showed increased CD40, CD86 and MHC11 activation markers on mDCs. Addition of matched HD naïve T cells 24 h post mDC-PDO coculture led to the excessive activation of CD4+ and CD8+ T cells highlighted by the PD1 expression on T cells from 0 h of initial T cell coculture to 48 h. This led us to treat PDO-mDC-T cell cocultures at 0 h to 48 h of initial T cell coculture with the anti-PD1 monoclonal antibody, Pembrolizumab. The treatment response was measured by live imaging of PDO growth followed by the analysis of changes in PDO diameter/area from day 0 of PDP to day 7 post-PDP. There were significant decreases in PDO growth in cultures treated with PDP followed by Pembrolizumab at different time points compared to untreated PDOs, untreated PDO-mDC-T cell cocultures, PDOs treated with PDP alone, PDO-mDC-T cell cocultures treated with PDP only or PDO-mDC-T cell cocultures treated with anti-PD1 alone. Also, we sorted T cells from the same cocultures at day 7 of PDP for gene expression analysis of T cell activation markers. There was enhanced expression of IFNγ, TNFα, FAS ligand, Granzyme B, IL2 and IL21 in T cells collected from combination therapy treated cocultures compared to others. Lastly, our data show that PDP can enhance anti-PD1 immunotherapy responses providing insights into a combination therapy option to treat PDAC. Citation Format: Pushpamali De Silva, Demi Wekking, Joanna Joeun Choe, Dario Missael Castellanos, Piotr Zelga, Russell Jenkins, Andrew Scott Liss, Tayyaba Hasan. Photodynamic priming enhances immunotherapy responses overcoming oncogenic drivers in pancreatic patient derived tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3228.

Photodynamic Priming Overcomes Per- and Polyfluoroalkyl Substance (PFAS)-Induced Platinum Resistance in Ovarian Cancer†.

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants linked to adverse outcomes, including for female reproductive biology and related cancers. We recently reported, for the first time, that PFAS induce platinum resistance in ovarian cancer, potentially through altered mitochondrial function. Platinum resistance is a major barrier in the management of ovarian cancer, necessitating complementary therapeutic approaches. Photodynamic therapy (PDT) is a light-based treatment modality that reverses platinum resistance and synergizes with platinum-based chemotherapy. The present study is the first to demonstrate the ability of photodynamic priming (PDP), a low-dose, sub-cytotoxic variant of PDT, to overcome PFAS-induced platinum resistance. Comparative studies of PDP efficacy using either benzoporphyrin derivative (BPD) or 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) were conducted in two human ovarian cancer cell lines (NIH:OVCAR-3 and Caov-3). BPD and PpIX are clinically approved photosensitizers that preferentially localize to, or are partly synthesized in, mitochondria. PDP overcomes carboplatin resistance in PFAS-exposed ovarian cancer cells, demonstrating the feasibility of this approach to target the deleterious effects of environmental contaminants. Decreased survival fraction in PDP + carboplatin treated cells was accompanied by decreased mitochondrial membrane potential, suggesting that PDP modulates the mitochondrial membrane, reducing membrane potential and re-sensitizing ovarian cancer cells to carboplatin.

Spotlight on Photoactivatable Liposomes beyond Drug Delivery: An Enabler of Multitargeting of Molecular Pathways.

The potential of photoactivating certain molecules, photosensitizers (PS), resulting in photochemical processes, has long been realized in the form of photodynamic therapy (PDT) for the management of several cancerous and noncancerous pathologies. With an improved understanding of the photoactivation process and its broader implications, efforts are being made to exploit the various facets of photoactivation, PDT, and the associated phenomenon of photodynamic priming in enhancing treatment outcomes, specifically in cancer therapeutics. The parallel emergence of nanomedicine, specifically liposome-based nanoformulations, and the convergence of the two fields of liposome-based drug delivery and PDT have led to the development of unique hybrid systems, which combine the exciting features of liposomes with adequate complementation through the photoactivation process. While initially liposomes carrying photosensitizers (PSs) were developed for enhancing the pharmacokinetics and the general applicability of PSs, more recently, PS-loaded liposomes, apart from their utility in PDT, have found several applications including enhanced targeting of drugs, coloading multiple therapeutic agents to enhance synergistic effects, imaging, priming, triggering drug release, and facilitating the escape of therapeutic agents from the endolysosomal complex. This review discusses the design strategies, potential, and unique attributes of these hybrid systems, with not only photoactivation as an attribute but also the ability to encapsulate multiple agents for imaging, biomodulation, priming, and therapy referred to as photoactivatable multiagent/inhibitor liposomes (PMILS) and their targeted versions─targeted PMILS (TPMILS). While liposomes have formed their own niche in nanotechnology and nanomedicine with several clinically approved formulations, we try to highlight how using PS-loaded liposomes could address some of the limitations and concerns usually associated with liposomes to overcome them and enhance their preclinical and clinical utility in the future.

Priming effect with photoinactivation against extensively drug-resistant Enterobacter cloacae and Klebsiella pneumoniae.

In this study, we present antimicrobial blue light (aBL) and antimicrobial photoinactivation with green light in the presence of Rose Bengal (aPDI) to modulate the susceptibility of extensively drug-resistant (XDR) Enterobacter cloacae and Klebsiella pneumoniae clinical isolates to antimicrobials. This process can be considered a photodynamic priming tool that influences other therapeutic options, such as antibiotics. The current study evaluated the different environments to estimate the most effective priming conditions by testing a broad spectrum of antimicrobials (including antimicrobials with different targets and mechanisms of action). The susceptibility of the E. cloacae and K. pneumoniae clinical isolates to various antibiotics after aBL and green light (with rose bengal) as aPDI treatment was examined with multiple methods of synergy testing (e.g., diffusion methods, checkerboard assay, postantibiotic effect), and most effective photoinactivation conditions were implemented for each environment. When Enterobacteriaceae were exposed to aBL, the most efficient reduction in survival rate under TSB conditions was observed. Similar results were observed when rose bengal, as a photosensitizer, was present during the exposure to green light in PBS. aBL and aPDI led to an increased susceptibility of K. pneumoniae and E. cloacae isolates to chloramphenicol and colistin or fosfomycin and colistin antibiotics, respectively. However, among the 4 tested isolates, we observed synergies between different antimicrobial agents and photoinactivation conditions. Thus, it may suggest that the sensitization process may be considered a strain dependent priming tool.

Increasing cancer permeability by photodynamic priming: from microenvironment to mechanotransduction signaling.

The dense cancer microenvironment is a significant barrier that limits the penetration of anticancer agents, thereby restraining the efficacy of molecular and nanoscale cancer therapeutics. Developing new strategies to enhance the permeability of cancer tissues is of major interest to overcome treatment resistance. Nonetheless, early strategies based on small molecule inhibitors or matrix-degrading enzymes have led to disappointing clinical outcomes by causing increased chemotherapy toxicity and promoting disease progression. In recent years, photodynamic therapy (PDT) has emerged as a novel approach to increase the permeability of cancer tissues. By producing excessive amounts of reactive oxygen species selectively in the cancer microenvironment, PDT increases the accumulation, penetration depth, and efficacy of chemotherapeutics. Importantly, the increased cancer permeability has not been associated to increased metastasis formation. In this review, we provide novel insights into the mechanisms by which this effect, called photodynamic priming, can increase cancer permeability without promoting cell migration and dissemination. This review demonstrates that PDT oxidizes and degrades extracellular matrix proteins, reduces the capacity of cancer cells to adhere to the altered matrix, and interferes with mechanotransduction pathways that promote cancer cell migration and differentiation. Significant knowledge gaps are identified regarding the involvement of critical signaling pathways, and to which extent these events are influenced by the complicated PDT dosimetry. Addressing these knowledge gaps will be vital to further develop PDT as an adjuvant approach to improve cancer permeability, demonstrate the safety and efficacy of this priming approach, and render more cancer patients eligible to receive life-extending treatments.

Towards Photodynamic Image-Guided Surgery of Head and Neck Tumors: Photodynamic Priming Improves Delivery and Diagnostic Accuracy of Cetuximab-IRDye800CW.

Fluorescence image-guided surgery (IGS) using antibody conjugates of the fluorophore IRDye800CW have revolutionized the surgical debulking of tumors. Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, conjugated to IRDye800CW (Cet-IRDye800) is the first molecular targeted antibody probe to be used for IGS in head and neck cancer patients. In addition to surgical debulking, Cetuximab-targeted photodynamic therapy (photoimmunotherapy; PIT) is emerging in the clinic as a powerful modality for head and neck tumor photodestruction. A plethora of other photoactivable agents are also in clinical trials for photodynamic-based therapies of head and neck cancer. Considering the vascular and stromal modulating effects of sub-therapeutic photodynamic therapy, namely photodynamic priming (PDP), this study explores the potential synergy between PDP and IGS for a novel photodynamic image-guided surgery (P-IGS) strategy. To the best of our knowledge, this is the first demonstration that PDP of the tumor microenvironment can augment the tumor delivery of full-length antibodies, namely Cet-IRDye800. In this study, we demonstrate a proof-of-concept that PDP primes orthotopic FaDu human head and neck tumors in mice for P-IGS by increasing the delivery of Cet-IRDye800 by up to 138.6%, by expediting its interstitial accumulation by 10.5-fold, and by increasing its fractional tumor coverage by 49.5% at 1 h following Cet-IRDye800 administration. Importantly, PDP improves the diagnostic accuracy of tumor detection by up to 264.2% with respect to vicinal salivary glands at 1 h. As such, PDP provides a time-to-surgery benefit by reducing the time to plateau 10-fold from 25.7 h to 2.5 h. We therefore propose that a pre-operative PDP regimen can expedite and augment the accuracy of IGS-mediated surgical debulking of head and neck tumors and reduce the time-to-IGS. Furthermore, this P-IGS regimen, can also enable a forward-looking post-operative protocol for the photodestruction of unresectable microscopic disease in the surgical bed. Beyond this scope, the role of PDP in the homogenous delivery of diagnostic, theranostic and therapeutic antibodies in solid tumors is of considerable significance to the wider community.

Abstract P031: Enrichment of photodynamically-primed anti-tumor immune infiltrates in pancreatic cancer: Enabling enhanced immunotherapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of 10%. It poorly responds to conventional cancer treatments. Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. However, in PDAC, even the modest success of ICB is limited to ~1-3% of patients as the majority of patients' tumors are considered as immunologically ‘'cold'' due to their highly immunosuppressive tumor microenvironment (TME). Photodynamic therapy (PDT) is an FDA approved anti-cancer therapy that utilizes light, a photoresponsive non-toxic chemical called a photosensitizer, and oxygen to generate reactive molecular species that confer direct cytotoxicity or vascular shutdown. PDT alters the TME transiently in a process termed photodynamic priming (PDP), making it more receptive to subsequent therapies, including chemo- and immunotherapy. Previous studies have demonstrated that PDP is capable of affecting both the innate and adaptive immune systems. These immune-stimulatory effects occur through its ability to induce immunogenic cell death via the release of damage-associated molecules (DAMPs) and tumor-associated antigens. In this study, we investigated PDP-induced immunogenicity in PDAC. In an immunocompetent mouse model of PDAC, we evaluated tumor-infiltrating lymphocyte (TIL) enrichment in tumors and ongoing immune responses in mouse spleens/blood from 1h to 120h post-PDP treatment comparing the responses in untreated controls. We observed gradual increases in T and B cell infiltration from 1h to 120h post-PDP where the T cell subset analysis showed an enrichment of CD8+ T cells in PDP-treated tumors. These CD8+ T cells showed temporal increases in PD1, CTLA4 and TIM3 immune checkpoints suggesting PDP-induced immune priming in the TME. This was further evidenced by the upregulation of DAMPs, including high mobility group box protein-1 and calreticulin in PDP treated tumors. Analyzing spleens of mice, we detected a significant increase in CD11C+MHC11hi dendritic cells from 1h to 24h post-PDP. Also, activation of an adaptive immune response in splenic B cell follicles was noted by the presence of proliferating germinal centers by 120h post-PDP. In addition, evaluating the blood of PDP treated mice, we detected expansion of CD8+ effector memory T and natural killer cell populations, signifying PDP-induced systemic immune responses compared to untreated mice. We further investigated how immune cells infiltrate PDP-treated tumors. A reduced formation of blood (CD31+) and lymphatic (Lyve-1+) vessels post-PDP at 120h was observed. However, decreases in PDL1, collagen and fibroblast activation proteins were observed in PDP-treated tumors at 120h, suggesting mitigation of immunosuppressive mechanisms and enhanced tumor permeability, allowing TIL migration. Our data shows converting immunologically silent PDAC tumors into inflamed ‘'hot'' tumors by triggering not only a local immune infiltration but also enhanced systemic immune responses, ultimately enhances the immunogenicity of pancreatic tumors. Citation Format: Pushpamali De Silva, Mohammad Ahsan Saad, Zhiming Mai, Shazia Bano, Assiris P. Camargo, Tayyaba Hasan. Enrichment of photodynamically-primed anti-tumor immune infiltrates in pancreatic cancer: Enabling enhanced immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P031.

180P Photodynamic priming of pancreatic cancer: Enabling immunotherapy

Pancreatic ductal adenocarcinoma (PDAC) poorly responds to cancer treatments including immunotherapy mainly due to the immunosuppressive tumor microenvironment (TME) it possesses. Photodynamic therapy (PDT) is an anti-cancer therapy that utilizes light, a photoresponsive non-toxic photosensitizer, and oxygen to generate reactive molecular species that confer cytotoxicity. PDT alters the TME transiently in a process termed photodynamic priming (PDP), making it more receptive to subsequent therapies.

Photodynamic Priming Improves the Anti-Migratory Activity of Prostaglandin E Receptor 4 Antagonist in Cancer Cells In Vitro.

Simple SummaryPhotodynamic priming is an emerging strategy that leverages subtherapeutic photochemistry for therapeutic benefits, often used as part of combination regimens. Our study aimed to couple photodynamically priming with antagonism of the prostaglandin E receptor 4, a therapeutic target linked to cancer-associated migration, invasion, angiogenesis, and immune evasion. Photodynamic priming and antagonism of the prostaglandin E receptor 4 independently attenuated OVCAR-5 ovarian cancer cell migration in a gap closure model, though their combination induced the most significant reductions. More potent combination effects were revealed when invasiveness was characterized using a transwell invasion model with CAOV3 ovarian cancer cells. Immunoblotting identified the epithelial growth factor receptor, cAMP-response element binding protein, and extracellular signal-regulated kinase 1/2 as potential mediators of these combinational effects. This work provides new evidence of a novel and clinically relevant combination strategy to address metastatic behavior, a major challenge in the treatment of cancer.The combination of photodynamic agents and biological inhibitors is rapidly gaining attention for its promise and approval in treating advanced cancer. The activity of photodynamic treatment is mainly governed by the formation of reactive oxygen species upon light activation of photosensitizers. Exposure to reactive oxygen species above a threshold dose can induce cellular damage and cancer cell death, while the surviving cancer cells are “photodynamically primed”, or sensitized, to respond better to other drugs and biological treatments. Here, we report a new combination regimen of photodynamic priming (PDP) and prostaglandin E2 receptor 4 (EP4) inhibition that reduces the migration and invasion of two human ovarian cancer cell lines (OVCAR-5 and CAOV3) in vitro. PDP is achieved by red light activation of the FDA-approved photosensitizer, benzoporphyrin derivative (BPD), or a chemical conjugate composed of the BPD linked to cetuximab, an anti-epithelial growth factor receptor (EGFR) antibody. Immunoblotting data identify co-inhibition of EGFR, cAMP-response element binding protein (CREB), and extracellular signal-regulated kinase 1/2 (ERK1/2) as key in the signaling cascades modulated by the combination of EGFR-targeted PDP and EP4 inhibition. This study provides valuable insights into the development of a molecular-targeted photochemical strategy to improve the anti-metastatic effects of EP4 receptor antagonists.

CT radiomic features of photodynamic priming in clinical pancreatic adenocarcinoma treatment

Photodynamic therapy (PDT) offers localized focal ablation in unresectable pancreatic tumors while tissues surrounding the treatment volume experience a lower light dose, termed photodynamic priming (PDP). While PDP does not cause tissue damage, it has been demonstrated to promote vascular permeability, improve drug delivery, alleviate tumor cell density, and reduce desmoplasia and the resultant internal pressure in pre-clinical evaluation. Preclinical data supports PDP as a neoadjuvant therapy beneficial to subsequent chemotherapy or immunotherapy, yet it is challenging to quantify PDP effects in clinical treatment without additional imaging and testing. This study investigated the potential of radiomic analysis using CT scans acquired before and after PDT to identify areas experiencing PDT-induced necrosis as well as quantify PDP effects in the surrounding tissues. A total of 235 CT tumor slices from seven patients undergoing PDT for pancreatic tumors were examined. Radiomic features assessed included intensity metrics (CT number in Hounsfield Units) and texture analysis using several gray-level co-occurrence matrix (GLCM) parameters. Pre-treatment scans of tumor areas that resulted in PDT-induced necrosis showed statistically significant differences in intensity and texture-based features that could be used to predict the regions that did respond (paired t-test, response versus no response, p < 0.001). Evaluation of PDP effects on the surrounding tissues also demonstrated statistically significant differences, in tumor mean value, standard deviation, and GLCM parameters of contrast, dissimilarity and homogeneity (t-test, pre versus post, p < 0.001). Using leave-one-out cross validation, six intensity and texture-based features were combined into a support-vector machine model which demonstrated reliable prediction of treatment effects for six out of seven patients (ROC curve, AUC = 0.93). This study provides pilot evidence that texture features extracted from CT scans could be utilized as an effective clinical diagnostic prediction and assessment of PDT and PDP effects in pancreatic tumors. (clinical trial NCT03033225)