PDT has shown clinical promise for the treatment of disseminated OvCa (14
), and will likely be most effective as part of a multifaceted treatment strategy to overcome resistance mechanisms that lead to treatment failure. The data presented in this study demonstrate a treatment-order dependent synergism with BPD-PDT and low-dose carboplatin using a 3D high-throughput reporter for adherent ovarian micrometastases.
Mechanistic differences between the individual modalities could account for the sequence-dependent synergism. Carboplatin is hydrolyzed as it enters a cell, creating an active species that forms interstrand and intrastrand DNA adducts. Depending on the extent of damage, the cell either enters cell cycle arrest or undergoes apoptosis, probably via ATM-CHK2 mediated activation of p53 in the nucleus (8
). This triggers transcriptional upregulation of pro-apoptotic proteins Bax/Bak and downregulation of anti-apoptotic Bcl-2/Bcl-XL
in the cytosol, causing permeabilization of the outer mitochondrial membrane. Cytochrome c
is subsequently released from the mitochondria followed by activation of caspase 9 and effector caspases 3,6 and 7, which triggers the apoptotic machinery that is responsible for DNA fragmentation and protein degradation typical of programmed cell death (8
In contrast, BPD-PDT bypasses the nuclear signaling pathways that platinum agents rely on by damaging the mitochondrial membrane and initiating cytochrome c
-mediated apoptosis or by directly destroying Bcl-2 associated with the mitochondria or endoplasmic reticulum (ER) (4
). The observed synergism between the two modalities could, therefore, be explained by a combination of three possible mechanisms: 1) BPD-PDT is in itself cytotoxic to target cells and decreases the size of residual ovarian tumors. As recently quantified by Celli et al. (36
), BPD-PDT shifts OvCa size distribution towards smaller nodules, in contrast to carboplatin, which had minimal impact on size reduction. These results could have important implications for designing more effective therapeutic regimens, since small OvCa nodules are associated with significantly better PFS, OS and response to chemotherapy than large tumors (5
). 2) BPD-PDT also disrupts nodular architecture creating tumors that are more vulnerable to carboplatin. High cellular density is a critical barrier to the penetration, and accumulation, of chemotherapeutic agents in tumors (7
). Treatment-induced apoptosis has specifically been shown to decrease cellular density and enhance the uptake of chemotherapies into tumor micronodules (7
). Therefore, BPD-PDT mediated apoptotic disruption of micronodular architecture could play an important role in improving the delivery of platinum-based agents into residual OvCa nodules. This enhanced diffusion is particularly important within the context of intraperitoneal administration of carboplatin, which relies on surface penetration of the drug to achieve therapeutic benefit (41
). 3) At the sub-cellular level, BPD-PDT sensitizes surviving cells to nuclear apoptotic signaling initiated by carboplatin, thereby lowering the threshold required to achieve a cytotoxic effect. The sequence-dependent synergism could therefore be driven by the ability of BPD-PDT to reduce the size and disrupt the structure of ovarian micronodules, in addition to sensitizing the cells to apoptotic signals from carboplatin treatment. Additional studies are necessary to elucidate the mechanisms for the observed synergism and to cross-validate these findings with tumor regrowth in 3D as well as focused in vivo
and patient tissue experiments.
The applicability of these findings to a broader library of photosensitizers and chemotherapies needs to considered within the context of cytotoxic mechanisms. PDT-induced cellular damage can trigger a combination of non-specific necrosis, apoptosis, or autophagy (18
). The predominance of a particular cytotoxic pathway depends on a variety of factors including the photophysical properties and preferred localization (and re-localization) sites of a photosensitizer, the local microenvironment, the fluence rate and PDT-dose, as well as compensatory survival mechanisms (18
). BPD, mesochlorin, and aluminum (III) phthalocyanine tetrasulfonate chloride (AlPcS(4)) localize primarily to the mitochondria, and are efficient inducers of apoptosis (24
). A more complex response is observed with photosensitizers that localize to the ER such as AlPcCl, and 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO) (18
). Depending on the PDT-dose, autophagy can be induced as either a pro-survival or pro-death pathway, which could be important in cells with defective apoptotic machinery, or resistance to apoptotis, and should be considered in mechanism-based combination regimens.
A variety of photosensitizers have been studied for PDT-mediated potentiation of chemotherapeutic agents including Photofrin, Photofrin II, delta-aminolevulinic acid (5-ALA), mesochlorin e6
monoethylene diamine (Mce6
), meta-tetra(hydroxyphenyl)chlorin (m-THPC, Foscan), and indocyanine green (ICG) (21
). The chemotherapeutic agents evaluated were cisplatin, doxorubicin and mytomicin C, which mediate cell death via DNA adducts (21
). PDT in combination with these pharmacological therapies was shown to enhance tumor destruction and reduce toxicity compared to chemotherapy alone (21
). The optimal treatment sequence, however, was dependent on the photosensitizer and chemotherapeutic agents that were used. Cisplatin cytotoxicity was enhanced most significantly when the chemotherapeutic was administered before Photofrin or ICG-based PDT. Similarly, the strongest potentiation of mitomycin C efficacy was seen when the drug was delivered prior to Photofrin II or 5-ALA-mediated PDT. Conversely, doxorubicin was most effective after treatment with Photofrin II, Mce6
or m-THPC-based PDT.
The in vitro
3D platform for micrometastatic OvCa described here fills a critical niche in translational science by bridging the gap between resource-intensive animal models and traditional monolayer cultures that lack important determinants of tumor growth and treatment response. Consistent with previous findings (12
), our results indicate that traditional monolayer cultures significantly overestimate the sensitivity of OvCa cells to cytotoxic treatments, which limits their value as tools to evaluate therapeutic efficacy. In contrast, tumor reduction in the same cells grown in 3D culture was comparable to results from in vivo
), which demonstrated that, as in the present study, multiple rounds of BPD-PDT or rationally-designed combinations were necessary to achieve significant therapeutic benefit. An additional strength of the 3D platform is the demonstrated ability to evaluate dosing schedules over a time period that more closely mimics in vivo
experiments than monolayer cultures. High resolution longitudinal imaging of cytotoxicity in the 3D platform also reveals differential cytotoxic patterns for carboplatin and BPD-PDT on a nodule-by-nodule basis that would be impossible to uncover in monolayer. These capabilities, combined with a system for high-throughput screening, facilitate rapid optimization of treatment parameters and allow valuable resources for in vivo
and patient tissue studies to be focused on the most promising regimens.
The treatment response factors addressed by this system include interaction of the monotherapies at the sub-cellular level and architectural disruption of ovarian nodules by BPD-PDT suggesting improved delivery of carboplatin. Future models will incorporate more complex aspects of the tumor microenvironment including co-cultures with stromal partners including fibroblasts and endothelial cells. These co-cultures are motivated in part by clinical findings from Menon et. al. (45
), who showed that nodules as small as 1mm (the smallest evaluated in the study) showed evidence of vascularity in peritoneal malignancies, including ovarian carcinomatosis. As the authors suggest, it was not clear if the vasculature in the smallest nodules was functional or even properly organized (45
). It is important to explore whether endothelial cells, as signaling partners, have an impact on tumor growth or treatment response, even in the absence of flow or structural organization. New in vitro
models for ovarian micrometastases using customizable matrices and stromal cells are being investigated by our group (46
) and others (47
), and will play an increasingly important role in screening combination regimens and uncovering resistance mechanisms.
Within the context of designing effective multifaceted treatments for OvCa, PDT-based combination regimens have been shown to reverse cisplatin resistance (13
), to synergistically increase the therapeutic effect of targeted biological therapies (4
), and now to synergistically enhance carboplatin efficacy for the treatment of multifocal OvCa. These findings are particularly significant in view of the fact that the highly toxic therapies currently used to treat metastatic OvCa have produced only modest improvements in the recurrence rates and mortality associated with this disease.
In an effort to address this challenge of a narrow therapeutic window, we used doses of BPD-PDT and carboplatin in this study that were substantially lower than the typical range used in vivo
and in the clinic (24
). Keeping in mind the need for well-tolerated treatments, tumor destruction could be further improved with incremental and concomitant increases in the doses of both monotherapies. This approach will likely be more successful than significantly increasing either monotherapy to higher and more toxic levels. Also, in contrast to carboplatin (23
), PDT can be administered in multiple rounds without additive host toxicity (4
). This attribute, along with PDT’s ability to sensitize chemoresistant and chemosensitive OvCa nodules to platinum-based agents (13
), highlights the need to explore the effect of repeatedly treating 3D micronodules with low-dose BPD-PDT in combination with low-dose carboplatin. The high throughput capabilities of the platform described here can be harnessed to evaluate these additional treatment scenarios, and to inform focused pre-clinical experiments.
The results and strategies presented here could be used to design more effective and well-tolerated clinical combination regimens, based on previously published studies using PDT to treat a variety of disseminated peritoneal malignancies, including ovarian carcinomatosis (14
). Phase I and II PDT clinical trials using non-optimized treatment parameters and Photofrin, have shown promise in treating cytoreduced minimal residual disease and chemoresistant peritoneal tumors (17
). Photofrin-PDT conferred a survival advantage relative to historic controls with acute but reversible toxicities. Furthermore, OvCas were among the most responsive intra-abdominal solid tumors to intraperitoneal PDT (17
). Building on these promising findings, we predict that BPD-PDT (which offers significant pharmacokinetic and photobiologic advantages over early generation photosensitizers)(4
) in combination with low-dose carboplatin will be an effective and well-tolerated combination regimen.
Collectively, these preclinical and clinical reports indicate that PDT should be included in the regular armamentarium used to treat ovarian carcinomatoses as part of a comprehensive treatment plan designed to minimize toxicity with optimal cytoreductive effects. We envision a scenario in which BPD-PDT is used in conjunction with cytoreductive surgery to treat and sensitize unresectable tumors to chemo-, radio- and biological therapeutics. Due to the vast library of candidate interventions, high-throughput 3D treatment response platforms, as presented here, will play a critical role in selecting the appropriate combination regimens for multifocal OvCa as well as many other lethal metastatic cancers.