Radiovaccination Strategy for Cancer Treatment Integrating Photodynamic Therapy-Generated Vaccines with Radiotherapy.

Therapeutic cancer vaccines have become firmly established as a reliable and proficient form of tumor immunotherapy. They represent a promising approach for substantial advancements in the successful treatment of malignant diseases. One attractive vaccine strategy is using, as the vaccine material, the whole tumor cells treated ex vivo by rapid tumor ablation therapies that instigate stress signaling responses culminating in immunogenic cell death (ICD). One such treatment is photodynamic therapy (PDT). The underlying mechanisms and critical elements responsible for the potency of these vaccines are discussed in this review. Radiotherapy has emerged as a suitable component for the combined therapy protocols with the vaccines. Arguments and prospects for optimizing tumor control using a radiovaccination strategy involving X-ray irradiation plus PDT vaccines are presented, together with the findings supporting its validity.

Mechanistic insights into ceramidase inhibitor LCL521-enhanced tumor cell killing by photodynamic and thermal ablation therapies.

Our recent investigation uncovered that the acid ceramidase inhibitor LCL521 enhances the direct tumor cell killing effect of photodynamic therapy (PDT) treatment. The present study aimed at elucidating the mechanisms underlying this effect. Exposing mouse squamous cell carcinoma SCCVII cells treated with temoporfin-based PDT to LCL521 (rising ceramide concentration) produced a much greater decrease in cell survival than comparable exposure to the sphingosine kinase-1 inhibitor PF543 (that reduces sphingosine-1-phosphate concentration). This is consistent with recognizing the rising levels of pro-apoptotic sphingolipid ceramide as being more critical in promoting the death of PDT-treated cells than the reduction in the availability of pro-survival acting sphingosine-1 phosphate. This pro-apoptotic impact of LCL521, which was suppressed by the apoptosis inhibitor bongkrekic acid, involves the interaction with the cellular stress signaling network. Hence, inhibiting the key elements of these pathways markedly influenced the adjuvant effect of LCL521 on the PDT response. Particularly effective was the inositol-requiring element-1 (IRE1) kinase inhibitor STF-083010 that dramatically enhanced the killing of cells treated with PDT plus LCL521. An important role in the survival of these cells was exhibited by master transcription factors STAT3 and HIF-1α. The STAT3 inhibitor NSC 74859 was especially effective in further reducing the cell survival rates, suggesting its possible exploitation for therapeutic gain. An additional finding in this study is that LCL521-promoted PDT-mediated cell killing through ceramide-mediated lethal effects is extended to the interaction with other cancer treatment modalities with a rapid cellular stress impact such as photothermal therapy (PTT) and cryoablation therapy (CAT).

Photodynamic therapy of cancer: an update.

Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. The procedure involves administration of a photosensitizing agent followed by irradiation at a wavelength corresponding to an absorbance band of the sensitizer. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature, and induction of a local inflammatory reaction. Clinical studies revealed that PDT can be curative, particularly in early stage tumors. It can prolong survival in patients with inoperable cancers and significantly improve quality of life. Minimal normal tissue toxicity, negligible systemic effects, greatly reduced long-term morbidity, lack of intrinsic or acquired resistance mechanisms, and excellent cosmetic as well as organ function-sparing effects of this treatment make it a valuable therapeutic option for combination treatments. With a number of recent technological improvements, PDT has the potential to become integrated into the mainstream of cancer treatment.

Role of cell stress signaling networks in cancer cell death and antitumor immune response following proteotoxic injury inflicted by photodynamic therapy.

The insult delivered by photodynamic therapy (PDT) in treated cells is oxidative stress. The main burden threatening survival of PDT-treated cells is proteotoxic damage that jeopardizes proteostasis in these cells. For dealing with this type of proteostasis impairment, cells have developed protection mechanisms operating by signaling networks. This review will outline various components of signaling networks that can be engaged in stressed cells with highlighting the emerging aspects relevant to response to PDT. It will be also shown how the well known inflammatory/immune response associated with PDT is also based on the activity of these stress signaling networks. Lasers Surg. Med. 50:491-498, 2018. © 2018 Wiley Periodicals, Inc.

Interaction of acid ceramidase inhibitor LCL521 with tumor response to photodynamic therapy and photodynamic therapy-generated vaccine.

Acid ceramidase has been identified as a promising target for cancer therapy. One of its most effective inhibitors, LCL521, was examined as adjuvant to photodynamic therapy (PDT) using mouse squamous cell carcinoma SCCVII model of head and neck cancer. Lethal effects of PDT, assessed by colony forming ability of in vitro treated SCCVII cells, were greatly enhanced when combined with 10 µM LCL521 treatment particularly when preceding PDT. When PDT-treated SCCVII cells are used to vaccinate SCCVII tumor-bearing mice (PDT vaccine protocol), adjuvant LCL521 treatment (75 mg/kg) resulted in a marked retardation of tumor growth. This effect can be attributed to the capacity of LCL521 to effectively restrict the activity of two main immunoregulatory cell populations (Tregs and myeloid-derived suppressor cells, MDSCs) that are known to hinder the efficacy of PDT vaccines. The therapeutic benefit with adjuvant LCL521 was also achieved with SCCVII tumors treated with standard PDT when using immunocompetent mice but not with immunodeficient hosts. The interaction of LCL521 with PDT-based antitumor mechanisms is dominated by immune system contribution that includes overriding the effects of immunoregulatory cells, but could also include a tacit contribution from boosting direct tumor cell kill.

The impact of macrophage-cancer cell interaction on the efficacy of photodynamic therapy.

Macrophages are one of the principal host cell populations in solid tumors. They are capable, due to their plasticity, of acquiring phenotypes that either combat (M1 type) or promote (M2 type) neoplastic growth. These cells, known as tumor-associated macrophages (TAMs), play complex but pivotal roles in the outcome of photodynamic therapy (PDT) of malignant lesions. Among the various parenchymal and stromal cell populations found in tumors, TAMs have been shown to have the greatest capacity for the uptake of systemically administered photosensitizers. Both the tumor-localizing property of photosensitizers and their tumor-localized fluorescence could be partly attributed to the activity of TAMs. Since resident TAMs with accumulated high photosensitizer content will sustain high degrees of PDT damage, this population (predominantly M2 in most tumors) is selectively destroyed, and during the ensuing inflammatory reaction is replaced with newly invading macrophages of M1 phenotype. These macrophages are sentinels responding to DAMP signals from PDT-treated tumor cells and in turn are mobilized to generate a variety of inflammatory/immune mediators and opsonins. They have a critical role in contributing to the therapeutic effect of PDT by mediating disposal of killed cancer cells and by processing/presenting tumor antigens to T lymphocytes. However, TAMs accumulating in the later post-PDT phase can acquire the M2 (healing) phenotype, and could have a role in tumor recurrence by releasing factors that promote angiogenesis and the survival/proliferation of remaining cancer cells. Various therapeutic strategies modulating TAM activity in the PDT response have potential for clinical use for improving PDT-mediated tumor control.

Immunoregulatory Cell Depletion Improves the Efficacy of Photodynamic Therapy-Generated Cancer Vaccines.

Photodynamic therapy (PDT)-generated cancer vaccine represents an attractive potential application of PDT, therapeutic modality destroying targeted lesions by localized photooxidative stress. Since immunoregulatory cell activity has become recognized as a major obstacle to effective cancer immunotherapy, the present study examined their participation in the therapeutic effect of PDT cancer vaccine. Following protocols from previous studies, mouse with squamous cell carcinoma SCCVII tumors were vaccinated by SCCVII cells treated by PDT and response monitored by tumor size measurement. The effects of low-dose cyclophosphamide (50 mg/kg) and all-trans retinoic acid (ATRA) on the numbers of Tregs and myeloid-derived suppressor cells (MDSCs) were determined by antibody staining followed by flow cytometry, while their impact on PDT vaccine therapy was evaluated by monitoring changes in tumor responses. Cyclophosphamide effectively reduced the numbers of Tregs, which became elevated following PDT vaccine treatment, and this resulted in an increase in the vaccine's effectiveness. A similar benefit for the therapy outcome with PDT vaccine was attained by ATRA treatment. The activities of Tregs and MDSCs thus have a critical impact on therapy outcome with PDT vaccine and reducing their numbers substantially improves the vaccine's effectiveness.

Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization.

The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.

Photodynamic Therapy Supported by Antitumor Lipids.

Photodynamic therapy (PDT) destroys tumors by generating cytotoxic oxidants that induce oxidative stress in targeted cancer cells. Antitumor lipids developed for cancer therapy act also by inflicting similar stress. The present study investigated whether tumor response to PDT can be improved by adjuvant treatment with such lipids using the prototype molecule edelfosine. Cellular stress intensity following Photofrin-based PDT, edelfosine treatment, or their combination was assessed by the expression of heat shock protein 70 (HSP70) on the surface of treated SCCVII tumor cells by FITC-conjugated anti-HSP70 antibody staining and flow cytometry. Surface HSP70 levels that became elevated after either PDT or edelfosine rose much higher after their combined treatment. The impact of Photofrin-PDT-plus-edelfosine treatment was studied with three types of tumor models grown in syngeneic mice. With both SCCVII squamous cell carcinomas and MCA205 fibrosarcoma, the greatest impact was with edelfosine peritumoral injection at 24 h after PDT, which substantially improved tumor cure rates. With Lewis lung carcinomas, edelfosine was highly effective in elevating PDT-mediated tumor cure rates even when injected peritumorally immediately after PDT. Edelfosine used before PDT was ineffective as adjuvant with all tumor models. The study findings provide proof-in-principle for use of cancer lipids with tumor PDT.

Photodynamic therapy-generated cancer vaccine elicits acute phase and hormonal response in treated mice.

Photodynamic therapy (PDT)-generated cancer vaccines have shown promising results in preclinical studies and are being introduced in the clinics. Using an SCCVII mouse squamous cell carcinoma-based whole-cell autologous PDT vaccine model developed in our previous work, we have examined systemic effects in vaccinated mice that could be related to the induction of acute phase response. The upregulation of gene encoding serum amyloid P component (prototypic mouse acute phase reactant) was detected in the liver and to a lesser degree in the tumor of vaccinated mice at 24 h post-PDT vaccine treatment. A strong upregulation of gene for heat shock protein 70 was found in both the liver and tumor of mice at 4 h after their PDT vaccine treatment. Changes in the expression of genes for glucocorticoid-induced leucine zipper and serum- and glucocorticoid-regulated kinase 1 that are highly responsive to glucocorticoid modulation were uncovered in both the tumor and liver of vaccinated mice. A rise in the levels of serum corticosterone was detected in mice at 24 h after PDT vaccine treatment. The results indicate that a sudden appearance of a large number of PDT vaccine cells elicits host responses for securing their optimized clearance, which in addition to producing seminal acute phase reactants includes the engagement of glucocorticoid hormones. It is becoming increasingly clear that a consummate execution of this process of PDT vaccine cell removal is critical for tumor antigen recognition and the attainment of potent antitumor immune response.

Monitoring ceramide and sphingosine-1-phosphate levels in cancer cells and macrophages from tumours treated by photodynamic therapy.

Eradication of tumours by photodynamic therapy (PDT) is accompanied by marked changes in local sphingolipid (SL) engagement. Because of the heterogeneity of cellular composition, analysis of tumour tissue homogenates to quantify SL species is inadequate for evaluating their levels in parenchymal cancer cell population. By staining tumour-derived single cell suspensions with antibodies specific to ceramide and sphingosine 1-phosphate (S1P) followed by flow cytometry, we were able to document changes in the levels of these two key SLs in cancer cells and tumour-associated macrophages (TAMs) of mouse SCCVII tumours following PDT. The results confirm previously obtained indications that tumour treatment by PDT induces a marked rise in ceramide levels in cancer cells within these lesions. Cancer cells from PDT-treated SCCVII tumours undergoing apoptosis were found to have much higher ceramide levels and substantially lower S1P levels than their viable counterparts. Compared to cancer cells, considerably higher ceramide and S1P levels were consistently found in TAMs. Treatment of SCCVII tumour-bearing mice with ceramide analog LCL29 induced a rise in ceramide levels in TAMs but not in cancer cells. When combined with PDT, LCL29 treatment produced a further increase in ceramide levels in TAMs while having no evident impact on ceramide content in cancer cells within same tumours. The results highlight SLs as important participants in tumour response to PDT and potential adjuvant therapeutic targets to PDT.

Controlling Immunoregulatory Cell Activity for Effective Photodynamic Therapy of Cancer.

Recently, it has become clear that a prerequisite requirement for most cancer therapies is controlling the negative impact of the activity of immunosuppressory cell populations. It is therefore of a considerable interest to develop treatments for containing the operation of major myeloid and lymphoid immunoregulatory cell populations. We have reported that acid ceramidase inhibitor LCL521 effectively overrides the activity of immunoregulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) engaged in the context of tumor response to photodynamic therapy (PDT). The present communication dissects and describes in detail the procedure for the use of LCL521 as an adjuvant to PDT for improved cure rates of treated tumors based on restricting the activity of immunoregulatory cell populations.

Involvement of damage-associated molecular patterns in tumor response to photodynamic therapy: surface expression of calreticulin and high-mobility group box-1 release.

Damage-associated molecular patterns (DAMPs), danger signal molecules expressed after injury or infection, have become recognized as prerequisite for orchestrating effective anti-tumor host response. The expression of two prototypical DAMPs, calreticulin and high-mobility group box-1 (HMGB1) protein, was examined following Photofrin-photodynamic therapy (PDT) of Lewis lung carcinoma (LLC) cells in vitro and LLC tumors growing in syngeneic mice. Cell surface expression of calreticulin was found to be highly increased at 1 h after PDT treatment both in vitro and in vivo. Increased exposure of calreticulin was also detected on the surface of macrophages from PDT-treated LLC tumors. At the same time interval, a rise in serum HMGB1 was detected in host mice. Intracellular staining of macrophages co-incubated for 16 h with PDT-treated LLC cells revealed elevated levels of HMGB1 in these cells. The knowledge of the involvement of these DAMPs uncovers important mechanistic insights into the development of host response induced by PDT.

Full range characterization of the Raman spectra of organs in a murine model.

Raman spectroscopy is a minimally-invasive optical technique with great potential for in vivo cancer detection and disease diagnosis. However, there is no systematic study of the Raman spectra from different organs to date. We measured and characterized the Raman spectra eighteen naïve mouse organs in a broad frequency range of 700 to 3100 cm⁻¹. The peaks of generic proteins and lipids appeared in Raman spectra of all organs. Some organs like bone, teeth, brain and lung had unique Raman peaks. The autofluorescence was strong in liver, spleen, heart, and kidney. These results suggest that organ specific Raman probe design and specific data processing strategies are required in order to get the most useful information.

Cancer vaccines generated by photodynamic therapy.

The development of photodynamic therapy (PDT)-generated cancer vaccines is potentially one of the most significant achievements in the field of PDT. Employing vaccine protocols optimizes the capacity of PDT of inducing a strong immune response against treated tumor due to the establishment of highly favourable conditions for maximizing the avidity of the immune reaction while sustaining and prolonging its tumor-destroying attack. While the introduction of PDT vaccines into the clinics and testing on patients is still in a very early phase, much work can still be done on further improvement of the potency of PDT vaccines. Considerable advances can be expected by identifying the most effective adjuvants to be used with PDT vaccines, which will most likely be different with different types of cancerous lesions.

Amplification of cancer cell apoptosis in photodynamic therapy-treated tumors by adjuvant ceramide analog LCL29.

BACKGROUND AND OBJECTIVES: C6-ceramide analog LCL29 was recently shown to improve the cure rates of mouse tumors treated by photodynamic therapy (PDT), but the mechanism underlying the therapeutic gain remains unclear. Since LCL29 is a pro-apoptotic agent, the main objective of the present study was to determine how LCL29 affects cancer cell apoptosis in PDT-treated tumors. STUDY DESIGN/MATERIALS AND METHODS: Mice bearing SCCVII tumors (syngeneic squamous cell carcinomas) were treated by PDT with photosensitizer Foscan. Adjuvant LCL29 treatment (40 mg/kg) was 24 hours before PDT. The tumors were excised 3 hours after PDT, disaggregated into single cell suspensions that were stained for flow cytometry to detect apoptosis-related events in cancer cells (caspase activation, loss of mitochondrial transmembrane potential, and intracellular calcium). In addition, phagocytic activity in tumor-associated macrophages was assessed by phalloidin staining. RESULTS: While 5-10% apoptotic cancer cells were found in tumors treated by PDT or LCL29 alone, close to 40% of such cells were found in tumors treated by LCL29 combined with PDT. Mitochondrial depolarization was detected in PDT, LCL29, and LCL29 + PDT groups, reaching similar values in all groups. Intracellular calcium release triggered by PDT was more pronounced when PDT was combined with LCL29. Macrophage phagocytic activity, negatively affected by PDT, was stimulated by adjuvant LCL29. CONCLUSIONS: Combining LCL29 treatment with PDT can enhance intracellular calcium release in cancer cells and strongly amplify their apoptosis.

Optimization of Whole Tumor Cell Vaccines by Interaction with Phagocytic Receptors.

The principal event in the function of whole-cell cancer vaccines is the ingestion of vaccine-delivered tumor antigen-containing material, which is performed by the patient's antigen-presenting cells (APCs) through the employment of their phagocytic receptors. The goal of the present study was to identify the phagocytic receptors critical for the therapeutic efficacy of whole-cell cancer vaccines. The model of photodynamic therapy (PDT)-generated vaccines based on mouse SCCVII tumors was utilized, with in vitro expanded SCCVII cells treated by PDT serving as the vaccine material used for treating mice bearing established SCCVII tumors. The therapeutic impact, monitored as delayed progression of vaccinated tumors, was almost completely eliminated when antibodies specifically blocking the activity of LOX-1 scavenger receptor were administered to mice 30 min before vaccination. Similar, but much less pronounced, impacts were found with antibodies neutralizing the activity of CR3/CR4 receptors recognizing complement-opsonized vaccine cells, and with those blocking activating Fcγ receptors that recognized IgG antibody-based opsonins. A strikingly contrary action, a greatly enhanced tumor control by the vaccine, was found by blocking immune inhibitory receptor, FcγRIIB. The reported findings establish, therefore, an attractive strategy that can be effectively exploited for potent therapeutic enhancement of PDT-generated (and probably other) whole-cell tumor vaccines.

Photodynamic Therapy as an Oxidative Anti-Tumor Modality: Negative Effects of Nitric Oxide on Treatment Efficacy.

Anti-tumor photodynamic therapy (PDT) is a unique oxidative stress-based modality that has proven highly effective on a variety of solid malignancies. PDT is minimally invasive and generates cytotoxic oxidants such as singlet molecular oxygen (1O2). With high tumor site-specificity and limited off-target negative effects, PDT is increasingly seen as an attractive alternative or follow-up to radiotherapy or chemotherapy. Nitric oxide (NO) is a short-lived bioactive free radical molecule that is exploited by many malignant tumors to promote cell survival, proliferation, and metastatic expansion. Typically generated endogenously by inducible nitric oxide synthase (iNOS/NOS2), low level NO can also antagonize many therapeutic interventions, including PDT. In addition to elevating resistance, iNOS-derived NO can stimulate growth and migratory aggressiveness of tumor cells that survive a PDT challenge. Moreover, NO from PDT-targeted cells in any given population is known to promote such aggressiveness in non-targeted counterparts (bystanders). Each of these negative responses to PDT and their possible underlying mechanisms will be discussed in this chapter. Promising pharmacologic approaches for mitigating these NO-mediated responses will also be discussed.

Novel Immune Stimulant Amplifies Direct Tumoricidal Effect of Cancer Ablation Therapies and Their Systemic Antitumor Immune Efficacy.

Ablation therapies have emerged as an effective tool for destroying cancerous tissue, but for advanced and disseminated tumors their application remains mainly a palliative measure. However, it is becoming increasingly clear that this limitation can be redressed by the use of intratumoral immune stimulating agents for amplifying potential antitumor immune responses that are induced by ablation therapies. A novel immune stimulating drug IP-001, a specific variant of the N-dihydrogalactochitosan (GC) family of molecules, has shown to be effective against metastatic tumors, when combined with different forms tumor ablation. It acts as a multi-function immune stimulant both by directly inhibiting cell membrane repair and recycling of ablation-damaged tumor cells, and indirectly by sequestering ablation-released tumor antigens, as well as recruiting and stimulating antigen presenting cells to induce a potent Th1 type T cell response against the cancer. In this review, we briefly discuss the current applications of local ablation for cancer treatment and the effects of GC in combination with other ablation therapies, a therapeutic approach that is pioneering the field of Interventional Immuno-Oncology (IIO).