Clinical assessment of a low-cost, hand-held, smartphone-attached intraoral imaging probe for 5-aminolevulinic acid photodynamic therapy monitoring and guidance.

Significance: India has one of the highest rates of oral squamous cell carcinoma (OSCC) in the world, with an incidence of 15 per 100,000 and more than 70,000 deaths per year. The problem is exacerbated by a lack of medical infrastructure and routine screening, especially in rural areas. New technologies for oral cancer detection and timely treatment at the point of care are urgently needed. Aim: Our study aimed to use a hand-held smartphone-coupled intraoral imaging device, previously investigated for autofluorescence (auto-FL) diagnostics adapted here for treatment guidance and monitoring photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence (FL). Approach: A total of 12 patients with 14 buccal mucosal lesions having moderately/well-differentiated micro-invasive OSCC lesions (<2 cm diameter and <5 mm depth) were systemically (in oral solution) administered three doses of 20 mg/kg ALA (total 60 mg/kg). Lesion site PpIX and auto-FL were imaged using the multichannel FL and polarized white-light oral cancer imaging probe before/after ALA administration and after light delivery (fractionated, total 100 J/cm2 of 635 nm red LED light). Results: The handheld device was conducive for access to lesion site images in the oral cavity. Segmentation of ratiometric images in which PpIX FL is mapped relative to auto-FL enabled improved demarcation of lesion boundaries relative to PpIX alone. A relative FL (R-value) threshold of 1.4 was found to segment lesion site PpIX production among the patients with mild to severe dysplasia malignancy. The segmented lesion size is well correlated with ultrasound findings. Lesions for which R-value was >1.65 at the time of treatment were associated with successful outcomes. Conclusion: These results indicate the utility of a low-cost, handheld intraoral imaging probe for image-guided PDT and treatment monitoring while also laying the groundwork for an integrated approach, combining cancer screening and treatment with the same hardware.

On the need for standardized reporting of photophysical parameters of in vitro photodynamic therapy studies.

In vitro dose escalation experiments are one of the first gatekeepers in therapeutic evaluation and development. This also holds for evaluating novel photosensitizers (PS) and Photodynamic Therapy (PDT) co-therapies as needed to provide dose response guidelines before engaging in further pre-clinical studies. The dose needed to achieve 50% cell kill (LD50) is a standard metric to report the potency of a therapeutic agents that is widely accepted for single-drug therapies. In reporting results of PDT experiments, which involve delivery of both drug and light, it is inherently more complicated to identify such a convenient dose response metric that actually captures the larger space of treatment parameters. In addition to ubiquitous sources of biological variability that apply broadly in biomedical research, PDT treatment efficacy is determined by multiple key parameters that may or may not have been documented, including PS concentration and light fluence, where the latter is itself a function of the spectral properties of the light source used (often not described), not to mention dose rate, fractionation and other parameters that potentially vary between individual studies. It is impossible to compare results between two study when, for example one reports LD50 PS concentration without providing essential light dosimetry details. Motivated by this challenge in comparing outcomes and establishing reproducibility of in vitro PDT studies, we endeavored to perform a meta-analysis of the reporting of PDT results by converting, where possible, the disparately reported experimental details into a consistent metric that could be used to compare across studies. In this context we adopt here the number of photons absorbed by photosensitizers per unit volume to affect a 50% decline in cell survival as a standardized metric. By choosing this metric one can acknowledge the quantum-based generation of cytotoxins. While this metric does not cover every possible source of variability between any two studies, for a PS with known optical properties, this does encapsulate PS concentration as well as irradiance and spectral properties of light delivered. For the sake of focus we adopt this approach for study of reported results with two photosensitizers, Protoporphyrin IX, either synthesized in the cells by aminolevulinic acid or administered exogenously, and Chlorin e6. A literature search was performed to identify in vitro studies with these two photosensitizers and collect necessary information to calculate the absorbed photon LD50 threshold for each study. Only approximately 1/10 of the manuscripts reporting on in vitro studies provide the minimum required information to calculate the threshold values. While the majority of the determined threshold values are within a factor of 10, the range of threshold values spanned close to 7 orders of magnitude for both photosensitizers. To contrast with single-agent therapies, a similar exercise was performed for chemotherapeutic drugs targeting cellular mitosis or tyrosine kinase inhibitors resulted in an LD50 or IC50 range of 1-2 orders of magnitude, with LD50 or IC50 values for a single cell line being within a factor of 5. This review underscores challenges in the reporting of in vitro PDT efficacy. In many cases it takes considerable effort to extract the necessary methodology information to make meaningful comparison between PDT studies. Only when results between studies can be compared is it possible to begin to assess reproducibility which, as shown here, can be a major issue. Hence, guidelines need to be developed and enforced through the peer review process for meaningful reporting of preclinical PDT results in order for the most promising sensitizers and co-therapies to be identified and translated into the clinic.

Photodynamic Stromal Depletion Enhances Therapeutic Nanoparticle Delivery in 3D Pancreatic Ductal Adenocarcinoma Tumor Models.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of human malignancies. PDAC is characterized by dense fibrous stroma which obstructs drug delivery and plays complex tumor-promoting roles. Photodynamic therapy (PDT) is a light-based modality which has been demonstrated to be clinically feasible and effective for tumors of the pancreas. Here, we use in vitro heterocellular 3D co-culture models in conjunction with imaging, bulk rheology and microrheology to investigate photodegradation of non-cellular components of PDAC stroma (photodynamic stromal depletion, PSD). By measuring the rheology of extracellular matrix (ECM) before and after PDT we find that softening of ECM is concomitant with increased transport of nanoparticles (NPs). At the same time, as shown by us previously, photodestruction of stromal fibroblasts leads to enhanced tumor response to PDT. Here we specifically evaluate the capability of PSD to enhance RNA nanomedicine delivery, using a NP carrying an inhibitor of miR-21-5P, a PDAC oncomiR. We confirm improved delivery of this therapeutic NP after PSD by observation of increased expression of PDCD4, a protein target of miR-21-5P. Collectively, these results in 3D tumor models suggest that PSD could be developed to enhance delivery of other cancer therapeutics and improve tumor response to treatment.

Clinical evaluation of a mobile, low-cost system for fluorescence guided photodynamic therapy of early oral cancer in India.

BACKGROUND: Morbidity and mortality due to oral cancer in India are exacerbated by a lack of access to effective treatments amongst medically underserved populations. We developed a user-friendly low-cost, portable fibre-coupled LED system for photodynamic therapy (PDT) of early oral lesions, using a smartphone fluorescence imaging device for treatment guidance, and 3D printed fibreoptic attachments for ergonomic intraoral light delivery. METHODS: 30 patients with T1N0M0 buccal mucosal cancer were recruited from the JN Medical College clinics, Aligarh, and rural screening camps. Tumour limits were defined by external ultrasound (US), white light photos and increased tumour fluorescence after oral administration of the photosensitising agent ALA (60 mg/kg, divided doses), monitored by a smartphone fluorescence imaging device. 100 J/cm2 LED light (635 nm peak) was delivered followed by repeat fluorescence to assess photobleaching. US and biopsy were repeated after 7-17 days. This trial is registered with ClinicalTrials.gov, NCT03638622, and the study has been completed. FINDINGS: There were no significant complications or discomfort. No sedation was required. No residual disease was detected in 22 out of 30 patients who completed the study (26 of 34 lesions, 76% complete tumour response, 50 weeks median follow-up) with up to 7.2 mm depth of necrosis. Treatment failures were attributed to large tumour size and/or inadequate light delivery (documented by limited photobleaching). Moderately differentiated lesions were more responsive than well-differentiated cancers. INTERPRETATION: This simple and low-cost adaptation of fluorescenceguided PDT is effective for treatment of early-stage malignant oral lesions and may have implications in global health.

Photodynamic Therapy for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of human cancers. Clinical trials of various chemotherapy, radiotherapy, targeted agents and combination strategies have generally failed to provide meaningful improvement in survival for patients with unresectable disease. Photodynamic therapy (PDT) is a photochemistry-based approach that enables selective cell killing using tumor-localizing agents activated by visible or near-infrared light. In recent years, clinical studies have demonstrated the technical feasibility of PDT for patients with locally advanced PDAC while a growing body of preclinical literature has shown that PDT can overcome drug resistance and target problematic and aggressive disease. Emerging evidence also suggests the ability of PDT to target PDAC stroma, which is known to act as both a barrier to drug delivery and a tumor-promoting signaling partner. Here, we review the literature which indicates an emergent role of PDT in clinical management of PDAC, including the potential for combination with other targeted agents and RNA medicine.

Photodestruction of Stromal Fibroblasts Enhances Tumor Response to PDT in 3D Pancreatic Cancer Coculture Models.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of human cancers. The dismal response of PDAC to virtually all therapeutics is associated, in part, with a characteristically dense fibrotic stroma. This stroma not only acts as a barrier to drug perfusion, but also promotes tumor survival through paracrine crosstalk and biophysical interactions. Photodynamic therapy (PDT) is being explored for PDAC treatment, though the impact of tumor-promoting stromal crosstalk on PDT response in PDAC is not well-characterized. The current study assesses the effect of tumor-stroma interactions on response to PDT or chemotherapy in heterocellular 3D cocultures using PDAC cells and two different fibroblastic cell types (pancreatic stellate cells, PSCs, and a normal human fibroblast cell line, MRC5) embedded in extracellular matrix (ECM). While stromal fibroblasts promote resistance to chemotherapy as expected, PDAC 3D nodules in coculture with fibroblasts exhibit increased response to PDT relative to homotypic cultures. These results point to the potential for PDT to overcome tumor-promoting stromal interactions associated with poor therapeutic response in PDAC.

Clinical evaluation of smartphone-based fluorescence imaging for guidance and monitoring of ALA-PDT treatment of early oral cancer.

SIGNIFICANCE: India has one of the highest rates of oral cancer incidence in the world, accounting for 30% of reported cancers. In rural areas, a lack of adequate medical infrastructure contributes to unchecked disease progression and dismal mortality rates. Photodynamic therapy (PDT) has emerged as an effective modality with potential for treating early stage disease in resource-limited settings, while photosensitizer fluorescence can be leveraged for treatment guidance. AIM: Our aim was to assess the capability of a simple smartphone-based device for imaging 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence for treatment guidance and monitoring as part of an ongoing clinical study evaluating low-cost technology for ALA-based PDT treatment of early oral cancer. APPROACH: A total of 29 subjects with <2 cm diameter moderately/well-differentiated microinvasive ( < 5 mm depth) oral squamous cell carcinoma lesions (33 lesions total, mean area ∼1.23 cm2) were administered 60 mg / kg ALA in oral solution and imaged before and after delivery of 100 J / cm2 total light dose to the lesion surface. Smartphone-based fluorescence and white light (WL) images were analyzed and compared with ultrasound (US) imaging of the same lesions. RESULTS: We present a comparative analysis of pre- and post-treatment fluorescence, WL, and US images of oral lesions. There was no significant difference in the distribution of lesion widths measured by fluorescence and US (mean widths of 14.5 and 15.3 mm, respectively) and linear regression shows good agreement (R2 = 0.91). In general, PpIX fluorescence images obtained prior to therapeutic light delivery are able to resolve lesion margins while dramatic photobleaching (∼42 % ) is visible post-treatment. Segmentation of the photobleached area confirms the boundaries of the irradiated zone. CONCLUSIONS: A simple smartphone-based approach for imaging oral lesions is shown to agree in most cases with US, suggesting that this approach may be a useful tool to aid in PDT treatment guidance and monitoring photobleaching as part of a low-cost platform for intraoral PDT.

Flow-induced Shear Stress Confers Resistance to Carboplatin in an Adherent Three-Dimensional Model for Ovarian Cancer: A Role for EGFR-Targeted Photoimmunotherapy Informed by Physical Stress.

A key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.

Photodynamic Therapy and the Biophysics of the Tumor Microenvironment.

Targeting the tumor microenvironment (TME) provides opportunities to modulate tumor physiology, enhance the delivery of therapeutic agents, impact immune response and overcome resistance. Photodynamic therapy (PDT) is a photochemistry-based, nonthermal modality that produces reactive molecular species at the site of light activation and is in the clinic for nononcologic and oncologic applications. The unique mechanisms and exquisite spatiotemporal control inherent to PDT enable selective modulation or destruction of the TME and cancer cells. Mechanical stress plays an important role in tumor growth and survival, with increasing implications for therapy design and drug delivery, but remains understudied in the context of PDT and PDT-based combinations. This review describes pharmacoengineering and bioengineering approaches in PDT to target cellular and noncellular components of the TME, as well as molecular targets on tumor and tumor-associated cells. Particular emphasis is placed on the role of mechanical stress in the context of targeted PDT regimens, and combinations, for primary and metastatic tumors.

Modulation of Extracellular Matrix Rigidity Via Riboflavin-mediated Photocrosslinking Regulates Invasive Motility and Treatment Response in a 3D Pancreatic Tumor Model.

In this study, we evaluate the use of riboflavin-mediated collagen photocrosslinking as an experimental tool to modulate extracellular matrix (ECM) mechanical properties in 3D in vitro tumor models. Using this approach in conjunction with 3D pancreatic tumor spheroid transplants, we show that the extent of matrix photocrosslinking in reconstituted hydrogels with fixed protein concentration scales inversely with the extent of invasive progression achieved by cells infiltrating into the surrounding ECM from primary transplanted spheroids. Using cross-linking to manipulate the extent of invasion into ECM in conjunction with imaging-based treatment assessment, we further leverage this approach as a means for assaying differential therapeutic response in primary nodule and ECM-invading populations and compare response to verteporfin-based photodynamic therapy (PDT) and oxaliplatin chemotherapy. Treatment response data shows that invading cell populations (which also exhibit markers of increased EMT) are highly chemoresistant yet have significantly increased sensitivity to PDT relative to the primary nodule. In contrast, the oxaliplatin treatment achieves greater growth inhibition of the primary nodule. These findings may be significant in themselves, while the methodology developed here could have a broader range of applications in developing strategies to target invasive disease and/or mecahanobiological determinants of therapeutic response in solid tumors.

Platform for ergonomic intraoral photodynamic therapy using low-cost, modular 3D-printed components: Design, comfort and clinical evaluation.

Oral cancer prevalence is increasing at an alarming rate worldwide, especially in developing countries which lack the medical infrastructure to manage it. For example, the oral cancer burden in India has been identified as a public health crisis. The high expense and logistical barriers to obtaining treatment with surgery, radiotherapy and chemotherapy often result in progression to unmanageable late stage disease with high morbidity. Even when curative, these approaches can be cosmetically and functionally disfiguring with extensive side effects. An alternate effective therapy for oral cancer is a light based spatially-targeted cytotoxic therapy called photodynamic therapy (PDT). Despite excellent healing of the oral mucosa in PDT, a lack of robust enabling technology for intraoral light delivery has limited its broader implementation. Leveraging advances in 3D printing, we have developed an intraoral light delivery system consisting of modular 3D printed light applicators with pre-calibrated dosimetry and mouth props that can be utilized to perform PDT in conscious subjects without the need of extensive infrastructure or manual positioning of an optical fiber. To evaluate the stability of the light applicators, we utilized an endoscope in lieu of the optical fiber to monitor motion in the fiducial markers. Here we showcase the stability (less than 2 mm deviation in both horizontal and vertical axis) and ergonomics of our applicators in delivering light precisely to the target location in ten healthy volunteers. We also demonstrate in five subjects with T1N0M0 oral lesions that our applicators coupled with a low-cost fiber coupled LED-based light source served as a complete platform for intraoral light delivery achieving complete tumor response with no residual disease at initial histopathology follow up in these patients. Overall, our approach potentiates PDT as a viable therapeutic option for early stage oral lesions that can be delivered in low resource settings.

Stochastic modeling of phenotypic switching and chemoresistance in cancer cell populations.

Phenotypic heterogeneity in cancer cells is widely observed and is often linked to drug resistance. In several cases, such heterogeneity in drug sensitivity of tumors is driven by stochastic and reversible acquisition of a drug tolerant phenotype by individual cells even in an isogenic population. Accumulating evidence further suggests that cell-fate transitions such as the epithelial to mesenchymal transition (EMT) are associated with drug resistance. In this study, we analyze stochastic models of phenotypic switching to provide a framework for analyzing cell-fate transitions such as EMT as a source of phenotypic variability in drug sensitivity. Motivated by our cell-culture based experimental observations connecting phenotypic switching in EMT and drug resistance, we analyze a coarse-grained model of phenotypic switching between two states in the presence of cytotoxic stress from chemotherapy. We derive analytical results for time-dependent probability distributions that provide insights into the rates of phenotypic switching and characterize initial phenotypic heterogeneity of cancer cells. The results obtained can also shed light on fundamental questions relating to adaptation and selection scenarios in tumor response to cytotoxic therapy.

Protease Degradation of Protein Coronas and Its Impact on Cancer Cells and Drug Payload Release.

The effect of matrix metalloproteinases (MMPs) on preformed protein coronas around spherical gold nanoparticles (AuNPs) was studied. Protein coronas of different compositions (human serum, human serum albumin, and collagen IV) were formed around AuNPs and characterized. The protease MMP-9 had different effects on the corona depending on the corona composition, resulting in different changes to the corona hydrodynamic diameter ( DH). When incubated with PANC-1 cells, the corona showed evidence of both increases as well as decreases in DH. Varying the composition of the corona influenced the MMP-9 activity. Furthermore, the corona was influenced not only by the protease activity of the MMP-9 but also by its ability to exchange with proteins in the preformed corona. This exchange could also occur with proteins in the media. Thus, the net effect of the MMP-9 was a combination of the MMP-9 protease activity and also exchange. Time scales for the exchange varied depending on the nature that make up the protein corona (weakly vs strongly bound corona proteins). Mass spectrometry was used to probe the protein corona composition and supported the exchange and degradation model. Together, these results indicate that the mechanism of protease activity on AuNP coronas involves both rearrangement and exchange, followed by degradation.

Development and evaluation of a low-cost, portable, LED-based device for PDT treatment of early-stage oral cancer in resource-limited settings.

BACKGROUND: Photodynamic therapy (PDT) using δ-aminolevulinic acid (ALA) photosensitization has shown promise in clinical studies for the treatment of early-stage oral malignancies with fewer potential side effects than traditional therapies. Light delivery to oral lesions can also carried out with limited medical infrastructure suggesting the potential for implementation of PDT in global health settings. OBJECTIVES: We sought to develop a cost-effective, battery-powered, fiber-coupled PDT system suitable for intraoral light delivery enabled by smartphone interface and embedded electronics for ease of operation. METHODS: Device performance was assessed in measurements of optical power output, spectral stability, and preclinical assessment of PDT response in ALA-photosensitized squamous carcinoma cell cultures and murine subcutaneous tumor xenografts. RESULTS: The system achieves target optoelectronic performance with a stable battery-powered output of approximately 180 mW from the fiber tip within the desired spectral window for PpIX activation. The device has a compact configuration, user friendly operation and flexible light delivery for the oral cavity. In cell culture, we show that the overall dose-response is consistent with established light sources and complete cell death of ALA photosensitized cells can be achieved in the irradiated zone. In vivo PDT response (reduction in tumor volume) is comparable with a commercial 635 nm laser. CONCLUSIONS: We developed a low-cost, LED-based, fiber-coupled PDT light delivery source that has stable output on battery power and suitable form factor for deployment in rural and/or resource-limited settings. Lasers Surg. Med. 9999:1-7, 2018. © 2018 Wiley Periodicals, Inc.

Neoadjuvant photodynamic therapy augments immediate and prolonged oxaliplatin efficacy in metastatic pancreatic cancer organoids.

Effective treatment of advanced metastatic disease remains the primary challenge in the management of inoperable pancreatic cancer. Current therapies such as oxaliplatin (OxPt)-based chemotherapy regimens (FOLFIRINOX) provide modest short-term survival improvements, yet with significant toxicity. Photodynamic therapy (PDT), a light-activated cancer therapy, demonstrated clinical promise for pancreatic cancer treatment and enhances conventional chemotherapies with non-overlapping toxicities. This study investigates the capacity of neoadjuvant PDT using a clinically-approved photosensitizer, benzoporphyrin derivative (BPD, verteporfin), to enhance OxPt efficacy in metastatic pancreatic cancer. Treatment effects were evaluated in organotypic three-dimensional (3D) cultures, clinically representative models that bridge the gap between conventional cell cultures and in vivo models. The temporally-spaced, multiparametric analyses demonstrated a superior efficacy for combined PDT+OxPt compared to each monotherapy alone, which was recapitulated on different organotypic pancreatic cancer cultures. The therapeutic benefit of neoadjuvant PDT to OxPt chemotherapy materialized in a time-dependent manner, reducing residual viable tissue and tumor viability in a manner not achievable with OxPt or PDT alone. These findings emphasize the need for intelligent combination therapies and relevant models to evaluate the temporal kinetics of interactions between mechanistically-distinct treatments and highlight the promise of PDT as a neoadjuvant treatment for disseminated pancreatic cancer.

ECM Composition and Rheology Regulate Growth, Motility, and Response to Photodynamic Therapy in 3D Models of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma is characterized by prominent stromal involvement, which plays complex roles in regulating tumor growth and therapeutic response. The extracellular matrix (ECM)-rich stroma associated with this disease has been implicated as a barrier to drug penetration, although stromal depletion strategies have had mixed clinical success. It remains less clear how interactions with ECM, acting as a biophysical regulator of phenotype, not only a barrier to drug perfusion, regulate susceptibilities and resistance to specific therapies. In this context, an integrative approach is used to evaluate invasive behavior and motility in rheologically characterized ECM as determinants of chemotherapy and photodynamic therapy (PDT) responses. We show that in 3D cultures with ECM conditions that promote invasive progression, response to PDT is markedly enhanced in the most motile ECM-infiltrating populations, whereas the same cells exhibit chemoresistance. Conversely, drug-resistant sublines with enhanced invasive potential were generated to compare differential treatment response in identical ECM conditions, monitored by particle tracking microrheology measurements of matrix remodeling. In both scenarios, ECM-infiltrating cell populations exhibit increased sensitivity to PDT, whether invasion is consequent to selection of chemoresistance, or whether chemoresistance is correlated with acquisition of invasive behavior. However, while ECM-invading, chemoresistant cells exhibit mesenchymal phenotype, induction of EMT in monolayers without ECM was not sufficient to enhance PDT sensitivity, yet does impart chemoresistance as expected. In addition to containing platform development with broader applicability to inform microenvironment-dependent therapeutics, these results reveal the efficacy of PDT for targeting the most aggressive, chemoresistant, invasive pancreatic ductal adenocarcinoma associated with dismal outcomes for this disease. IMPLICATIONS: ECM-infiltrating and chemoresistant pancreatic tumor populations exhibit increased sensitivity to PDT. Mol Cancer Res; 15(1); 15-25. ©2016 AACR.

Low-cost photodynamic therapy devices for global health settings: Characterization of battery-powered LED performance and smartphone imaging in 3D tumor models.

A lack of access to effective cancer therapeutics in resource-limited settings is implicated in global cancer health disparities between developed and developing countries. Photodynamic therapy (PDT) is a light-based treatment modality that has exhibited safety and efficacy in the clinic using wavelengths and irradiances achievable with light-emitting diodes (LEDs) operated on battery power. Here we assess low-cost enabling technology to extend the clinical benefit of PDT to regions with little or no access to electricity or medical infrastructure. We demonstrate the efficacy of a device based on a 635 nm high-output LED powered by three AA disposable alkaline batteries, to achieve strong cytotoxic response in monolayer and 3D cultures of A431 squamous carcinoma cells following photosensitization by administering aminolevulinic acid (ALA) to induce the accumulation of protoporphyrin IX (PpIX). Here we characterize challenges of battery-operated device performance, including battery drain and voltage stability specifically over relevant PDT dose parameters. Further motivated by the well-established capacity of PDT photosensitizers to serve as tumour-selective fluorescence contrast agents, we demonstrate the capability of a consumer smartphone with low-cost add-ons to measure concentration-dependent PpIX fluorescence. This study lays the groundwork for the on-going development of image-guided ALA-PDT treatment technologies for global health applications.

Digital holographic microscopy for longitudinal volumetric imaging of growth and treatment response in three-dimensional tumor models.

We report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, nondestructive longitudinal imaging of in vitro three-dimensional (3-D) tumor models. Following established methods, we prepared 3-D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple time points throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify the nodule thickness over time under normal growth and in cultures subject to chemotherapy treatment. In this manner, total nodule volumes are rapidly estimated and demonstrated here to show contrasting time-dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3-D structure over time and suggests the further development of this approach for time-lapse monitoring of 3-D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.

Longitudinal measurement of extracellular matrix rigidity in 3D tumor models using particle-tracking microrheology.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models.

While it is increasingly recognized that three-dimensional (3D) cell culture models recapitulate drug responses of human cancers with more fidelity than monolayer cultures, a lack of quantitative analysis methods limit their implementation for reliable and routine assessment of emerging therapies. Here, we introduce an approach based on computational analysis of fluorescence image data to provide high-content readouts of dose-dependent cytotoxicity, growth inhibition, treatment-induced architectural changes and size-dependent response in 3D tumour models. We demonstrate this approach in adherent 3D ovarian and pancreatic multiwell extracellular matrix tumour overlays subjected to a panel of clinically relevant cytotoxic modalities and appropriately designed controls for reliable quantification of fluorescence signal. This streamlined methodology reads out the high density of information embedded in 3D culture systems, while maintaining a level of speed and efficiency traditionally achieved with global colorimetric reporters in order to facilitate broader implementation of 3D tumour models in therapeutic screening.