Corrigendum: Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

[This corrects the article DOI: 10.3389/fimmu.2023.1278000.].

Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

Background: Immune-checkpoint inhibitors (ICIs) represent a revolution in cancer therapy and are currently implemented as standard therapy within several cancer indications. Nevertheless, the treatment is only effective in a subset of patients, and immune-related adverse effects complicate the improved survival. Adjuvant treatments that can improve the efficacy of ICIs are highly warranted, not only to increase the response rate, but also to reduce the therapeutic ICI dosage. Several treatment modalities have been suggested as ICI adjuvants including vascular targeted treatments and photodynamic therapy (PDT). Photochemical internalization (PCI) is a drug delivery system, based on PDT. PCI is long known to generate an immune response in murine models and was recently shown to enhance the cellular immune response of a vaccine in a clinical study. In the present work we evaluated PCI in combination with the vascular targeting toxin VEGF121/rGel with respect to induction of immune-mediated cell death as well as in vitro ICI enhancement. Methods: DAMP signaling post VEGF121/rGel-PCI was assessed in CT26 and MC38 murine colon cancer cell lines. Hypericin-PDT, previously indicated as an highly efficient DAMP inducer (but difficult to utilize clinically), was used as a control. ATP release was detected by a bioluminescent kit while HMGB1 and HSP90 relocalization and secretion was detected by fluorescence microscopy and western blotting. VEGF121/rGel-PCI was further investigated as an αCTLA enhancer in CT26 and MC38 tumors by measurement of tumor growth delay. CD8+ Dependent efficacy was evaluated in vivo using a CD8+ antibody. Results: VEGF121/rGel-PCI was shown to induce increased DAMP signaling as compared to PDT and VEGF121/rGel alone and the magnitude was found similar to that induced by Hypericin-PDT. Furthermore, a significant CD8+ dependent enhanced αCTLA-4 treatment effect was observed when VEGF121/rGel-PCI was used as an adjuvant in both tumor models. Conclusions: VEGF121/rGel-PCI describes a novel concept for ICI enhancement which induces a rapid CD8+ dependent tumor eradication in both CT26 and MC38 tumors. The concept is based on the combination of intracellular ROS generation and vascular targeting using a plant derived toxin and will be developed towards clinical utilization.

RAB5A expression is a predictive biomarker for trastuzumab emtansine in breast cancer.

HER2 is a predictive biomarker for HER2-targeted therapeutics. For antibody-drug conjugates (ADCs; e.g., trastuzumab emtansine (T-DM1)), HER2 is utilized as a transport gate for cytotoxic agents into the cell. ADC biomarkers may therefore be more complex, also reflecting the intracellular drug transport. Here we report on a positive correlation between the early endosome marker RAB5A and T-DM1 sensitivity in five HER2-positive cell lines. Correlation between RAB5A expression and T-DM1 sensitivity is confirmed in breast cancer patients treated with trastuzumab emtansine/pertuzumab in the I-SPY2 trial (NCT01042379), but not in the trastuzumab/paclitaxel control arm. The clinical correlation is further verified in patients from the KAMILLA trial (NCT01702571). In conclusion, our results suggest RAB5A as a predictive biomarker for T-DM1 response and outline proteins involved in endocytic trafficking as predictive biomarkers for ADCs.

Inhibiting autophagy increases the efficacy of low-dose photodynamic therapy.

Rupture and permeabilization of endocytic vesicles can be triggered by various causes, such as pathogenic invasions, amyloid proteins, and silica crystals leading to cell death and degeneration. A cellular quality control process, called lysophagy was recently described to target damaged lysosomes for autophagic sequestration within isolation membranes in order to protect the cell from the consequences of lysosomal leakage. This protective process, however, might interfere with treatment conditions, such as photodynamic therapy (PDT) and the intracellular drug delivery method photochemical internalization (PCI). PCI-induced permeabilization of endosomes and lysosomes is purposely triggered to release drugs that are sequestered in these organelles into the cytosol in order to synergistically kill cancer cells. Here, we show that photochemical treatment with the PCI-photosensitizer TPCS2a/fimaporfin results in both induction of autophagy and inhibition of the autophagic flux. The autophagic response is accompanied by recruitment of ubiquitin (Ubq), p62, and microtubule-associated protein 1A/1B-light chain 3 (LC3) to damaged vesicles, marked by Galectin 3 (Gal3). Furthermore, ultrastructural analysis revealed a homogenously thick p62-positive layer surrounding these permeabilized vesicles. Although p62 seems to be important during the selective autophagic sequestration, we show that its presence is not essential for the effective removal of damaged vesicles or the recovery of the lysosomal content. An active autophagic response and the presence of p62, however, is important for cancer cells to survive low-dose TPCS2a-PDT. Thus, targeting both p62 and autophagy together and independently, in a light-controlled/PCI based delivery of cancer therapeutics could increase the effectiveness of the treatment regime.

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System.

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05-0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

Photochemically-Induced Release of Lysosomal Sequestered Sunitinib: Obstacles for Therapeutic Efficacy.

Lysosomal accumulation of sunitinib has been suggested as an underlying mechanism of resistance. Here, we investigated if photochemical internalization (PCI), a technology for cytosolic release of drugs entrapped in endosomes and lysosomes, would activate lysosomal sequestered sunitinib. By super-resolution fluorescence microscopy, sunitinib was found to accumulate in the membrane of endo/lysosomal compartments together with the photosensitizer disulfonated tetraphenylchlorin (TPCS2a). Furthermore, the treatment effect was potentiated by PCI in the human HT-29 and the mouse CT26.WT colon cancer cell lines. The cytotoxic outcome of sunitinib-PCI was, however, highly dependent on the treatment protocol. Thus, neoadjuvant PCI inhibited lysosomal accumulation of sunitinib. PCI also inhibited lysosomal sequestering of sunitinib in HT29/SR cells with acquired sunitinib resistance, but did not reverse the resistance. The mechanism of acquired sunitinib resistance in HT29/SR cells was therefore not related to lysosomal sequestering. Sunitinib-PCI was further evaluated on HT-29 xenografts in athymic mice, but was found to induce only a minor effect on tumor growth delay. In immunocompetent mice sunitinib-PCI enhanced areas of treatment-induced necrosis compared to the monotherapy groups. However, the tumor growth was not delayed, and decreased infiltration of CD3-positive T cells was indicated as a possible mechanism behind the failed overall response.

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research.

Photochemical internalisation (PCI) is a unique intervention which involves the release of endocytosed macromolecules into the cytoplasmic matrix. PCI is based on the use of photosensitizers placed in endocytic vesicles that, following light activation, lead to rupture of the endocytic vesicles and the release of the macromolecules into the cytoplasmic matrix. This technology has been shown to improve the biological activity of a number of macromolecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus and oligonucleotides and certain chemotherapeutics, such as bleomycin. This new intervention has also been found appealing for intracellular delivery of drugs incorporated into nanocarriers and for cancer vaccination. PCI is currently being evaluated in clinical trials. Data from the first-in-human phase I clinical trial as well as an update on the development of the PCI technology towards clinical practice is presented here.

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization.

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Light-enhanced VEGF121/rGel: A tumor targeted modality with vascular and immune-mediated efficacy.

Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4 T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.

Photochemical delivery of bleomycin induces T-cell activation of importance for curative effect and systemic anti-tumor immunity.

Photochemical internalization (PCI) is a technology to enhance intracellular drug delivery by light-induced translocation of endocytosed therapeutics into the cytosol. The aim of this study was to explore the efficacy of PCI-based delivery of bleomycin and the impact on systemic anti-tumor immunity. Mouse colon carcinoma cells (CT26.CL25), stably expressing the bacterial ß-galactosidase, were inoculated into the legs of athymic or immuno-competent BALB/c mice strains. The mice were injected with the photosensitizer AlPcS2a and bleomycin (BLM) prior to tumor light exposure from a 670nm diode laser. Photochemical activation of BLM was found to induce synergistic inhibition of tumor growth as compared to the sum of the individual treatments. However, a curative effect was not observed in the athymic mice exposed to 30J/cm2 of light while >90% of the thymic mice were cured after exposure to only 15J/cm2 light. Cured thymic mice, re-challenged with CT26.CL25 tumor cells on the contralateral leg, rejected 57-100% of the tumor cells inoculated immediately and up to 2months after the photochemical treatment. T-cells from the spleen of PCI-treated mice were found to inhibit the growth of CT26.CL25 cells in naïve thymic mice with a 60% rejection rate. The results show that treatment of CT26.CL25 tumors in thymic mice by PCI of BLM induces a systemic anti-tumor immunity.

Development of resistance to photodynamic therapy (PDT) in human breast cancer cells is photosensitizer-dependent: Possible mechanisms and approaches for overcoming PDT-resistance.

Here we report on the induction of resistance to photodynamic therapy (PDT) in the ABCG2-high human breast cancer cell line MA11 after repetitive PDT, using either Pheophorbide A (PhA) or di-sulphonated meso-tetraphenylchlorin (TPCS2a) as photosensitizer. Resistance to PhA-PDT was associated with enhanced expression of the efflux pump ABCG2. TPCS2a-PDT-resistance was neither found to correspond with lower TPCS2a-accumulation nor reduced generation of reactive oxygen species (ROS). Cross-resistance to chemotherapy (doxorubicin) or radiotherapy was not observed. TPCS2a-PDT-resistant cells acquired a higher proliferation capacity and an enhanced expression of EGFR and ERK1/2. p38 MAPK was found to be a death-signalling pathway in the MA11 cells post TPCS2a-PDT, contrasting the MA11/TR cells in which PDT generated a sustained phosphorylation of p38 that had lost its death-mediated signalling, and an abrogated activation of its downstream effector MAPKAPK2. No difference in apoptosis, necrosis or autophagy responses was found between the treated cell lines. Development of TPCS2a-PDT resistance in the MDA-MB-231 cell line was also established, however, p38 MAPK did not play a role in the PDT-resistance. MCF-7 cells did not develop TPCS2a-PDT-resistance. Photochemical internalisation (PCI) of 1 pM of EGF-saporin induced equal strong cytotoxicity in both MA11 and MA11/TR cells. In conclusion, loss of p38 MAPK-inducing death signalling is the main mechanism of resistance to TPCS2a-PDT in the MA11/TR cell line. This work provides mechanistic knowledge of intrinsic and acquired PDT-resistance which is dependent on choice of photosensitizer, and suggests PCI as a rational therapeutic intervention for the elimination of PDT-resistant cells.

Photochemical activation of MH3-B1/rGel: a HER2-targeted treatment approach for ovarian cancer.

HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson's correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson's correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer.

Photochemical activation of drugs for the treatment of therapy-resistant cancers.

Resistance to chemotherapy, molecular targeted therapy as well as radiation therapy is a major obstacle for cancer treatment. Cancer resistance may be exerted through multiple different mechanisms which may be orchestrated as observed in multidrug resistance (MDR). Cancer resistance may be intrinsic or acquired and often leaves patients without any treatment options. Strategies for alternative treatment modalities for resistant cancer are therefore highly warranted. Photochemical internalization (PCI) is a technology for cytosolic delivery of macromolecular therapeutics based on the principles of photodynamic therapy (PDT). The present report reviews the current knowledge of PCI of therapy-resistant cancers. In summary, PCI may be able to circumvent several of the major mechanisms associated with resistance towards chemotherapeutics including increased expression of drug efflux pumps, altered intracellular drug distribution and increased ROS scavenging. Current data also suggest PCI of targeted toxins as highly effective in cancers resistant to clinically available targeted therapy such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs). PCI may therefore, in general, represent a future treatment option for cancers resistant to other therapies.

Photochemical internalisation, a minimally invasive strategy for light-controlled endosomal escape of cancer stem cell-targeting therapeutics.

Despite progress in radio-, chemo- and photodynamic-therapy (PDT) of cancer, treatment resistance still remains a major problem for patients with aggressive tumours. Cancer stem cells (CSCs) or tumour-initiating cells are intrinsically and notoriously resistant to conventional cancer therapies and are proposed to be responsible for the recurrence of tumours after therapy. According to the CSC hypothesis, it is imperative to develop novel anticancer agents or therapeutic strategies that take into account the biology and role of CSCs. The present review outlines our recent study on photochemical internalisation (PCI) using the clinically relevant photosensitiser TPCS2a/Amphinex® as a rational, non-invasive strategy for the light-controlled endosomal escape of CSC-targeting drugs. PCI is an intracellular drug delivery method based on light-induced ROS-generation and a subsequent membrane-disruption of endocytic vesicles, leading to cytosolic release of the entrapped drugs of interest. In different proof-of-concept studies we have demonstrated that PCI of CSC-directed immunotoxins targeting CD133, CD44, CSPG4 and EpCAM is a highly specific and effective strategy for killing cancer cells and CSCs. CSCs overexpressing CD133 are PDT-resistant; however, this is circumvented by PCI of CD133-targeting immunotoxins. In view of the fact that TPCS2a is not a substrate of the efflux pumps ABCG2 and P-glycoprotein (ABCB1), the PCI-method is a promising anti-CSC therapeutic strategy. Due to a laser-controlled exposure, PCI of CSC-targeting drugs will be confined exclusively to the tumour tissue, suggesting that this drug delivery method has the potential to spare distant normal stem cells.

Light-triggered, efficient cytosolic release of IM7-saporin targeting the putative cancer stem cell marker CD44 by photochemical internalization.

We have used the site specific and light-depended drug delivery method photochemical internalization (PCI) to release an immunotoxin (IT), targeting the CD44 receptor, into the cytosol of target cells. The IT consisted of a pan CD44 mAb (clone IM7) bound to the ribosome inactivating protein (RIP) saporin by a biotin-streptavidin linker named IM7-saporin. PCI is based upon photosensitizing compounds localized in the membrane of endosomes and lysosomes causing membrane rupture upon illumination followed by release of the IT into the cytosol. In this in vitro study, we have used 7 different human cancer cell lines of various origins to investigate the cytotoxic effect of PCI-based targeting of the cancer stem cell (CSC) marker CD44. Epi-fluorescence microscopy shows both specific binding and uptake of the IM7-Alexa488, after 30 min and 18 h of incubation, and colocalization with the PCI-photosensitizer TPCS2a prior to light-triggered cytosolic release of the CD44-targeting IT. PCI of IM7-saporin resulted in efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells. A higher level of reactive oxygen species (ROS) was found in untreated and photodynamic therapy (PDT)-treated LNCaP (CD44(neg)) compared to that of DU145 (CD44(pos)) prostate cancer (PC) cells. This may explain the PDT-resistance observed in the DU145 cells. PCI-based targeting of CD44-expressing cancer cells gives very potent and specific cytotoxic effects and may represent a rational strategy for achieving site-selective elimination of CSCs in aggressive androgen-independent and treatment-resistant PC cells preventing cytotoxic effects on distant normal stem cells.

Photochemical activation of the recombinant HER2-targeted fusion toxin MH3-B1/rGel; Impact of HER2 expression on treatment outcome.

HER2 is overexpressed in 20-30% of breast tumors and is associated with aggressiveness and increased risk of recurrence and death. The HER2 protein is internalized as a part of its activity, and may therefore be utilized as a target for the specific intracellular delivery of drugs. Photochemical internalization (PCI) is a novel technology now undergoing clinical evaluation for its ability to improve the release into the cytosol of drugs entrapped in the endo/lysosomal compartment. PCI employs an amphiphilic photosensitizer which localizes in the membranes of endo/lysosomes. Subsequent light exposure (visible light) causes destabilization of the endo/lysosomal membranes. PCI has been proven highly effective for improving the cytosolic delivery of targeted toxins based on type I ribosome inactivating protein toxins such as gelonin. We examined the impact of the level of target antigen expression on PCI efficacy. Four human breast cancer cell lines (MDA-MB-231, BT-20, Zr-75-1 and SK-BR-3) covering a wide range of HER2 expression were included in the present study. PCI of the HER2-targeted fusion toxin MH3-B1/rGel was found to be highly effective in all four cell lines. The increase in PCI-mediated efficacy was not directly correlated with the cellular levels of HER2 as assessed by western blots, the overall uptake of MH3-B1/rGel as measured by flow cytometry, the amount of MH3-B1/rGel localized to endo/lysosomes assessed by confocal microscopy or the cell sensitivity to the photochemical treatment itself (photosensitizer and light without MH3-B1/rGel). However, correcting the PCI efficacy for the baseline cellular sensitivity to rGel revealed a linear correlation (R(2)=0.80) with HER2 expression. The present report therefore concludes the cellular sensitivity to the toxin as an important parameter for PCI efficacy and also indicates PCI of a HER2-targeted fusion toxin as an attractive treatment alternative for breast cancer patients with both HER2-low and -high expression.

Photochemical internalization augments tumor vascular cytotoxicity and specificity of VEGF(121)/rGel fusion toxin.

Vascular targeting for cancer is increasingly recognized as a therapeutic strategy although the lack of objective responses and the development of resistance are major limitations for clinically-available drugs. Endothelial targeted toxins exert increased toxicity compared to antiangiogenic drugs and may therefore overcome these limitations. The specificity and toxicity of targeted toxins may be increased by utilization of a drug delivery system which provides selective release of the targeted toxins in the target cells. Photochemical internalization (PCI) is a non-invasive modality which causes translocation into the cytosol of agents that are trapped in endosomes. This study describes the first use of PCI in combination with a recombinant fusion toxin targeting tumor vasculature. Endothelial cells bearing VEGFR2 treated with VEGF121/rGel showed dramatic enhancement of toxicity after PCI utilizing the photosensitizer TPCS2a (Amphinex®). We compared the PCI of VEGF121/rGel to that of bleomycin which is currently under clinical evaluation. The VEGFR2 specificity of VEGF121/rGel was shown to be preserved by the PCI treatment. PCI of VEGF121/rGel was further shown to induce vascular collapse and edema in the invasive areas of CT26.CL25 colon carcinoma tumors as shown by CD31 IHC. Antitumor effects, as assessed by tumor growth delay were found for PCI of VEGF121/rGel and PCI of bleomycin with cure rates of 40% and 33% respectively. PCI of VEGF121/rGel was, however, better tolerated compared to PCI of bleomycin. Thus, PCI of vascular targeted toxins provides higher specificity and increased tolerability compared to PCI of bleomycin and may represent an interesting clinical future for the PCI technology.

Circumvention of resistance to photodynamic therapy in doxorubicin-resistant sarcoma by photochemical internalization of gelonin.

A wide range of anti-cancer therapies have been shown to induce resistance upon repetitive treatment and such adapted resistance may also cause cross-resistance to other treatment modalities. We here show that MES-SA/Dx5 cells with adapted resistance to doxorubicin (DOX) are cross-resistant to photodynamic therapy (PDT). A DOX-induced increased expression of the reactive oxygen species (ROS)-scavenging proteins glutathione peroxidase (GPx) 1 and GPx4 in MES-SA/Dx5 cells was indicated as the mechanism of resistance to PDT in line with the reduction in PDT-generated ROS observed in this cell line. ROS-induced p38 activation was, in addition, shown to be reduced to one-third of the signal of the parental MES-SA cells 2h after PDT, and addition of the p38 inhibitor SB203580 confirmed p38 activation as a death signal after PDT in the MES-SA cells. The MES-SA/Dx5 cells were also cross-resistant to ionizing radiation in agreement with the increased GPx1 and GPx4 expression. Surprisingly, PDT-induced endo/lysosomal release of the ribosome-inactivating protein gelonin (photochemical internalization (PCI)) was more effective in the PDT-resistant MES-SA/Dx5 cells, as measured by synergy calculations in both cell lines. Analysis of death-inducing signaling indicated a low activation of caspase-3 and a strong PARP I cleavage after PDT and PCI in both cell lines. The PARP I activation was, however, stronger after PCI than after PDT in the MES-SA cells, but not in the MES-SA/Dx5 cells, and therefore cannot explain the strong PCI effect in the MES-SA/Dx5 cells. In conclusion PCI of recombinant gelonin circumvents ROS resistance in an apoptosis-independent manner.

Vascular endothelial cells as targets for photochemical internalization (PCI).

Cancer treatment can be exerted by targeting both cancer cells and the vasculature supplying solid tumors. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, but recent data on contrast-enhanced MRI have indicated that the method also reduces blood perfusion in HT1080 fibrosarcoma xenografts. The present report aims to investigate if PCI may exert direct cytotoxic effects on endothelial cells. PCI of saporin was performed on endothelial human umbilical vein endothelial cell (HUVEC) and fibrosarcoma cells (HT1080) using two PCI-relevant photosensitizers, TPPS2a and AlPcS2a. A 22- and 13-fold higher photosensitizer uptake was detected in the endothelial cells compared with the HT1080 cells for AlPcS2a and TPPS2a, respectively. PCI of saporin was, however, found more effective in HT1080 cells. For HT1080 cells, PCI with saporin increased cell killing 1.9-fold over photodynamic therapy alone, but under the same conditions, only increased HUVEC cell killing by 1.6- and 1.3-fold with AlPcS2a and TPPS2a , respectively. Saporin uptake was higher in HUVECs than in the HT1080 cells, hence did not reflect the cell line differences in PCI efficacy. This is the first report on PCI-mediated kill of endothelial cells and lays the foundation for further preclinical evaluation of the PCI technology as an antivascular strategy to ablate tumors.

Sustained ERK [corrected] inhibition by EGFR targeting therapies is a predictive factor for synergistic cytotoxicity with PDT as neoadjuvant therapy.

BACKGROUND: Tyrosin kinase inhibitors (TKIs) and monoclonal antibodies aimed to target epidermal growth factor receptor (EGFR) have shown limited effect as monotherapies and drug resistance is a major limitation for therapeutic success. Adjuvant therapies to EGFR targeting therapeutics are therefore of high clinical relevance. METHODS: Three EGFR targeting drugs, Cetuximab, Erlotinib and Tyrphostin AG1478 were used in combination with photodynamic therapy (PDT) in two EGFR positive cell lines, A-431 epidermoid skin carcinoma and WiDr colorectal adenocarcinoma cells. The amphiphilic meso-tetraphenylporphine with 2 sulphonate groups on adjacent phenyl rings (TPPS(2a)) was utilized as a photosensitizer for PDT. The cytotoxic outcome of the combined treatments was evaluated by cell counting and MTT. Cellular signalling was explored by Western blotting. RESULTS: PDT as neoadjuvant to Tyrphostin in A-431 cells as well as to Tyrphostin or Erlotinib in WiDr cells revealed synergistic cytotoxicity. In contrast, Erlotinib or Cetuximab combined with neoadjuvant PDT induced an antagonistic effect on cell survival of A-431 cells. Neoadjuvant PDT and EGFR targeting therapies induced a synergistic inhibition of ERK as well as synergistic cytotoxicity only when the EGFR targeting monotherapies caused a prolonged ERK inhibition. There were no correlation between EGFR inhibition by the EGFR targeting monotherapies or the combined therapies and the cytotoxic outcome combination-therapies. CONCLUSIONS: The results suggest that sustained ERK inhibition by EGFR targeting monotherapies is a predictive factor for synergistic cytotoxicity when combined with neoadjuvant PDT. GENERAL SIGNIFICANCE: The present study provides a rationale for selecting anticancer drugs which may benefit from PDT as adjuvant therapy.