Mladen Korbelik

Mediators of peripheral blood neutrophilia induced by photodynamic therapy of solid tumors

Photodynamic therapy (PDT) of tumors elicits a strong host immune response and one of its manifestations is a pronounced neutrophilia. By blocking their function prior to Photofrin-based PDT of mouse EMT6 tumors, we have identified multiple mediators whose regulated action is responsible for this neutrophilia. In addition to complement fragments (direct mediators) released as a consequence of PDT-induced complement activation, there are at least a dozen secondary mediators that all arise as a result of complement activity. The latter include cytokines IL-1beta, TNF-alpha, IL-6, IL-10, G-CSF and KC, thromboxane, prostaglandins, leukotrienes, histamine, and coagulation factors.

Cancer treatment by photodynamic therapy combined with adoptive immunotherapy using genetically altered natural killer cell line

Adoptive immunotherapy mediated by human natural killer (NK) cell line genetically altered to produce interleukin‐2 (NK92MI) was evaluated as adjuvant to photodynamic therapy (PDT) of subcutaneous tumors. The combined effect of these two modalities was first examined with SiHa tumors (human cervical squamous cell carcinoma) growing in NOD‐scid mice. The most effective protocol for NK92MI cell transfer in conjunction with PDT mediated by photosensitizer mTHPC was the injection of 5 × 107 cells (peritumoral or intravenous) given immediately after PDT, which produced a marked improvement in the therapeutic outcome compared with the effect of PDT alone. The same protocol was tested with HT‐29 tumor model (human colorectal adenocarcinoma) xenografted in NOD‐scid mice. The results demonstrate that the adoptive immunotherapy with NK92MI cells (which when used alone were not effective in controlling tumor growth) significantly improved the cures of PDT‐treated HT‐29 tumors, whereas such benefit was not observed with the parental cell line NK92 (not producing interleukin‐2). Flow cytometry‐based analysis revealed a higher percentage of p.t. injected NK92MI cells in PDT‐treated than in non‐treated HT‐29 tumors. Further investigation showed that the NK92MI cell‐based adoptive immunotherapy is also a highly effective adjuvant for PDT treatment of murine EMT6 tumors growing in immunocompetent syngeneic BALB/c mice. This result diminishes the concern that adoptively transferred NK92MI cells may be rendered ineffective by an allogenic reaction of the host. The findings of this study suggest that advanced protocols of NK cell‐based adoptive immunotherapy can be developed as efficient adjuvants to PDT used for the treatment of solid malignant tumors.

THE EFFECT OF DIFFERENTIATION ON PHOTOSENSITIZER UPTAKE BY HL60 CELLS

The capability of human promyelocytic leukemia cells HL60 to be induced to differentiate to various stages along the monocytic or myelocytic pathway was exploited for investigation of the uptake of selected photo‐sensitizers by diverse types of cells of the same origin. The results showed that there was no substantial difference in photofrin uptake between noninduced HL60 cells, immature monocytes, immature neutrophils and cells differentiated along the eosinophilic pathway. In contrast, HL60 cells differentiated into macrophages (HL609) exhibited markedly increased photofrin uptake, which was further enhanced by their pretreatment with bacterial lipopolysaccharide. Similar results were obtained with other photosensitizers tested: di‐and tetrasulfonated aluminum phthalocyanines (AIPcS2 and AIPcS4), tetrasulfonated zinc phthalocyanine (ZnPcS4), tetraphenylporphine tetrasulfonate (TPPS4) and benzoporphyrin derivative monoacid (BPD). Despite marked differences in the state of self‐aggregation and other chemical properties of these compounds, the degree of their preferential uptake by HL60 PH cells showed very little variation. In a typical experiment, the uptake of these photosensitizers by HL60 PH cells was four to five times higher than the uptake by noninduced HL60 cells. In addition to the fluorometric assay employed in most of the experiments, cellular concentration of AlPcS4 was determined by measurement of elementary aluminum using atomic absorption spectroscopy.

Relevance of nitric oxide to the response of tumors to photodynamic therapy

Oxidative stress is the term used for a sudden and intense exposure of living tissue to reactive oxygen radicals. Tumor tissue response to oxidative stress, invoked in the action of photodynamic therapy (PDT) and some other modalities for cancer treatment, at the level of vascular endothelium has important therapeutic implications. Nitric oxide (NO), a transient radical species which is an important bioregulatory molecule involved in a diverse array of physiological events, has important functions in the regulation of progression of cancerous growth. Response to cancer therapies associated with the induction of oxidative stress was suggested to be amenable to NO mediation. Events involved in antitumor effects of PDT that can be markedly affected by changes in NO availability are listed. The correlation between endogenous NO production in tumors and the response of these lesions to PDT is discussed. Results of treatments aimed at modulating NO levels in PDT treated tumors are reviewed and evaluated.

Can PDT be potentiated by immunotherapy

Two principal aspects of interlinkage of the immune system with photodynamic therapy (PDT) treatment are discussed in detail: (1) participation of tumor associated macrophages (TAM) in tumor localization of photosensitizers, and (2) induction of an inflammatory immune response by phototoxic effects in endothelial cells, TAM and other cells in the tumor, leading to necrosis of the tumor tissue. It is illustrated how Photofrin levels in TAM, and consequently in the tumor as a whole, can be altered (increased or decreased) by specific agents that stimulate or impair physiological processes in TAM. It is suggested that the selectivity of tumor localization of some photosensitizers can be augmented by TAM targeted immunotherapy with agents that enhance the rate of tumor infiltration of these cells and stimulate their phagocytic activity. Preliminary results of ongoing studies suggest that PDT induces a massive infiltration of immune cells into the treated tumor. It is hypothesized that by combining PDT with an appropriate type of immunotherapy the immune reaction can be potentiated and directed against the surviving tumor cells. As as an example, it is shown that much better control of SCCVII tumor is achieved by combining PDT with the treatment by the immunoactivator SPG compared to PDT alone.

Microlocalization of Photofrin in neoplastic lesions

Three tumor models were used to study the microlocalization of two photosensitizers, Photofrin and TPPS4 (mesotetra[p-sulphophenyl]porphyne). The three tumor types employed were subcutaneously and intramuscularly grown murine Lewis lung carcinoma, and lung micrometastases, grown on the same animal. The objective of the study was to determine whether these photosensitizers localize differently in these three tumor types. Common to all the tumors was the observation that no substantial photosensitizer fluorescence could be found in the tumor parenchyma. With Photofrin, most of the drug fluorescence was found in the periphery of the tumors, especially around blood vessels and possibly in some macrophages. On the other hand with TPPS4, most of the fluorescence was seen in the basement membrane of blood vessels and airways. The photosensitizer TPPS4 may therefore accumulate less specifically in tumor microenvironment than Photofrin. Both photosensitizers were also found to localize in necrotic areas.

Modification of potentially lethal damage repair by some intrinsic intra- and extracellular agents: I. Proteinases and proteinase inhibitors.

The effects of nine intra- and extracellular proteinases and six proteinase inhibitors on the repair of potentially lethal damage (PLDR) induced by gamma-rays in plateau-phase V79 cells were examined. It was demonstrated that these agents, which are intrinsic factors produced within mammalian cells, can modify PLDR activity. A stimulatory effect on PLDR was seen with calf liver neutral proteinase, and to a lesser extent, with inhibitor pepstatin A. Other proteinases which belong to serine, cysteine and aspartic superfamilies, as well as proteinase inhibitors, inhibited PLDR to different degrees. The effects of some of these agents, present during the PLDR period, on the rate of tritiated thymidine incorporation into the acid-insoluble cell fraction was also examined. They can modify the DNA synthesis of cells when subcultured from plateau phase for the assessment of colony-forming ability. There is no clear evidence that the effects observed are entirely attributed to the alteration of cellular proliferative processes. It seems more likely that many serine and cysteine proteinases and their inhibitors can adversely affect the PLDR process by modulating the activity of proteinase(s) and other enzymes involved more directly in PLDR because of interrelationships of the entire intracellular proteinase system.

Role of inflammatory cytokines in the response of solid cancers to photodynamic therapy

Photodynamic therapy (PDT) elicits a strong acute inflammatory response that has both local and systemic (acute phase response) attributes. The insult mediated by PDT-induced oxidative stress at the targeted site triggers a complex multifactorial response engaging host defence mechanisms associated with the inflammatory process to participate in the eradication of the treated tumor. Inflammatory cytokines are important mediators of critical events in this process as they regulate the activity of inflammatory, endothelial and other cells. The initial stimulus for enhanced production and release of cytokines likely originates from several types of events, such as activated transcription factors and complement deposition. The PDT-induced complement activation appears to be directly linked to the enhanced expression of various cytokines, including chemokines such as KC (in mouse models), and classic inflammatory cytokines such as IL-1β, TNF-α , IL-6 and IL-10. A variety of interventions that modulate the activity of particular cytokines performed in conjunction with PDT were shown to influence the therapy outcome. The treatments such as using blocking antibodies and local or systemic cytokine delivery may either reduce or dramatically improve the curative effect of PDT. The inflammatory and related cytokines that at present appear particularly interesting and merit further investigation for use as adjuvants to PDT are IL-3, IL-8, IL-15, TNF-α, IFN-γ, G-CSF and GM-CSF.

Distribution of photosensitizers between tumor cells and tumor infiltrating host cells

Photofrin levels in different cellular populations constituting a murine FsaR fibrosarcoma were measured by flow cytometry. Both myeloid and lymphoid populations associated with the tumor were found to accumulate more photosensitizer on a per cell basis, on average, than the malignant cells. Macrophages, identified by the F4/80 antigen, exceeded other myeloid cells in Photofrin accumulation. It is shown that one of the factors involved is the variability in the photosensitizer content in cells located at different distances from the nearest blood vessel. This was investigated by a flow cytometry technique with the fluorescent stain Hoechst 33342, used to distinguish cells depending on their proximity to the tumor vasculature.

Modulation of tumor response to photodynamic therapy in severe combined immunodeficient (SCID) mice by adoptively transferred lymphoid cells

Photodynamic treatment, consisting of intravenous injection of PhotofrinR (10 mg/kg) followed by exposure to 110 J/cm2 of 630 plus or minus 10 nm light 24 hours later, cured 100% of EMT6 tumors (murine mammary sarcoma) growing in syngeneic immunocompetent BALB/C mice. In contrast, the same treatment produced no cures of EMT6 tumors growing in either nude or SCID mice (immunodeficient strains). EMT6 tumors growing in BALB/C and SCID mice showed no difference in either the level of PhotofrinR accumulated per gram of tumor tissue, or the extent of tumor cell killing during the first 24 hours post photodynamic therapy (PDT). In an attempt to improve the sensitivity to PDT of EMT6 tumors growing in SCID mice, these hosts were given either splenic T lymphocytes or whole bone marrow from BALB/C mice. The adoptive transfer of lymphocytes 9 days before PDT was successful in delaying tumor recurrence but produced no cures. A better improvement in PDT response was obtained with tumors growing in SCID mice reconstituted with BALB/C bone marrow (tumor cure rate of 63%). The results of this study demonstrate that, at least with the EMT6 tumor model, antitumor immune activity mediated by lymphoid cell populations makes an important contribution to the curative effect of PDT.