Anti-tumor immunity is stimulated after PDT due a number of factors including: the acute inflammatory response caused
by PDT, release of antigens from PDT-damaged tumor cells, priming of the adaptive immune system to recognize
tumor-associated antigens (TAA), and induction of heat-shock proteins. The induction of specific CD8+ T-lymphocyte
cells that recognize major histocompatibility complex class I (MHC-I) restricted epitopes of TAAs is a highly desirable
goal in cancer therapy as it would allow the treatment of tumors that may have already metastasized. The PDT killed
tumor cells may be phagocytosed by dendritic cells (DC) that then migrate to draining lymph nodes and prime naïve T-cells
that recognize TAA epitopes. We have carried out in vivo PDT with a BPD-mediated vascular regimen using a pair
of BALB/c mouse colon carcinomas: CT26 wild type expressing the naturally occurring retroviral antigen gp70 and
CT26.CL25 additionally expressing beta-galactosidase (b-gal) as a model tumor rejection antigen. PDT of CT26.CL25
cured 100% of tumors but none of the CT26WT tumors (all recurred). Cured CT26.CL25 mice were resistant to
rechallenge. Moreover mice with two bilateral CT26.CL25 tumors that had only one treated with PDT demonstrated
spontaneous regression of 70% of untreated contralateral tumors. T-lymphocytes were isolated from lymph nodes of
PDT cured mice that recognized a particular peptide specific to b-gal antigen. T-lymphocytes from LN were able to kill
CT26.CL25 target cells in vitro but not CT26WT cells as shown by a chromium release assay. CT26.CL25 tumors
treated with PDT and removed five days later had higher levels of Th1 cytokines than CT26 WT tumors showing a
higher level of immune response. When mice bearing CT26WT tumors were treated with a regimen of low dose
cyclophosphamide (CY) 2 days before, PDT led to 100% of cures (versus 0% without CY) and resistance to rechallenge.
Low dose CY is thought to deplete regulatory T-cells (Treg, CD4+CD25+foxp3+) and potentiate immune response after
PDT in the case of tumors that express self-antigens. These data suggest that PDT alone will stimulate a strong immune
response when tumors express a robust antigen, and in cases where tumors express a self-antigen, T-reg depletion can
unmask the immune response after PDT.
Photodynamic therapy (PDT) involves the administration of photosensitizers followed by illumination of the
primary tumor with red light producing reactive oxygen species that cause vascular shutdown and tumor cell
necrosis and apoptosis. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response,
priming of the immune system to recognize tumor-associated antigens (TAA). The induction of specific CD8+ Tlymphocyte
cells that recognize major histocompatibility complex class I (MHC-I) restricted epitopes of TAAs is a
highly desirable goal in cancer therapy. The PDT killed tumor cells may be phagocytosed by dendritic cells (DC)
that then migrate to draining lymph nodes and prime naïve T-cells that recognize TAA epitopes. This process is
however, often sub-optimal, in part due to tumor-induced DC dysfunction. Instead of DC that can become mature
and activated and have a potent antigen-presenting and immune stimulating phenotype, immature dendritic cells
(iDC) are often found in tumors and are part of an immunosuppressive milieu including regulatory T-cells and
immunosuppressive cytokines such as TGF-beta and IL10. We here report on the use of a potent DC activating
agent, an oligonucleotide (ODN) that contains a non-methylated CpG motif and acts as an agonist of toll like
receptor (TLR) 9. TLR activation is a danger signal to notify the immune system of the presence of invading
pathogens. CpG-ODN (but not scrambled non-CpG ODN) increased bone-marrow DC activation after exposure to
PDT-killed tumor cells, and significantly increased tumor response to PDT and mouse survival after peri-tumoral
administration. CpG may be a valuable immunoadjuvant to PDT especially for tumors that produce DC dysfunction.
It has been known for many years that low level laser (or light) therapy (LLLT) can ameliorate the pain, swelling
and inflammation associated with various forms of arthritis. Light is absorbed by mitochondrial chromophores
leading to an increase in ATP, reactive oxygen species and/or cyclic AMP production and consequent gene
transcription via activation of transcription factors. However, despite many reports about the positive effects of
LLLT in medicine, its use remains controversial. Our laboratory has developed animal models designed to
objectively quantify response to LLLT and compare different light delivery regimens. In the arthritis model we
inject zymosan into rat knee joints to induce inflammatory arthritis. We have compared illumination regimens
consisting of a high and low fluence (3 J/cm<sup>2</sup> and 30 J/cm<sup>2</sup>), delivered at a high and low irradiance (5 mW/cm2 and 50 mW/cm<sup>2</sup>) using 810-nm laser light daily for 5 days, with the effect of conventional corticosteroid
(dexamethasone) therapy. Results indicated that illumination with 810-nm laser is highly effective (almost as good
as dexamethasone) at reducing swelling and that longer illumination time was more important in determining
effectiveness than either total fluence delivered or irradiance. Experiments carried out using 810-nm LLLT on
excisional wound healing in mice also confirmed the importance of longer illumination times. These data will be of
value in designing clinical trials of LLLT.
Cancer is a leading cause of death among modern peoples largely due to metastatic disease. The ideal cancer
treatment should target both the primary tumor and the metastases with the minimal toxicity. This is best
accomplished by educating the body's immune system to recognize the tumor as foreign so that after the
primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves
the IV administration of photosensitizers followed by illumination of the primary tumor with red light
producing reactive oxygen species that cause vascular shutdown and tumor cell apoptosis. Anti-tumor
immunity is stimulated after PDT due to the acute inflammatory response, priming of the immune system to
recognize tumor-associated antigens (TAA), and induction of heat-shock proteins. The induction of specific
CD8+ T lymphocyte cells that recognize major histocompatibility complex class I (MHC-I) restricted
epitopes of TAAs is a highly desirable goal in cancer therapy. We here report on PDT of mice bearing
tumors that either do or do not express an established TAA. We utilized a BALB/c colon adenocarcinoma
cell line termed CT26.CL25 retrovirally transduced to stably express &bgr;-galactosidase ( &bgr;-gal, a bacterial
protein), and its non-&bgr;-gal expressing wild-type counterpart termed CT26 WT, as well as the control cell line
consisting of CT26 transduced with the empty retroviral vector termed CT26-neo. All cells expressed class I
MHC restriction element H-2Ld syngenic to BALB/c mice. Vascular PDT with a regimen of 1mg/kg BPD
injected IV, and 120 J/cm<sup>2</sup> of 690-nm laser light after 15 minutes successfully cured 100% of CT26.CL25
tumors but 0% of CT26-neo tumors and 0% of CT26 WT tumors. After 90 days tumor free interval the
CT26.CL25 cured mice were rechallenged with CT26.CL25 tumor cells and 96% rejected the rechallenge
while the CT26.CL25 cured mice did not reject a CT26 WT tumor cell challenge. Experiments with mice
bearing two CT26.CL25 tumors (one in each leg) and only one tumor treated with PDT, showed that the
immune response was strong enough to destroy an already established tumor in 70 of the mice and this effect
was not seen with mice bearing two CT26 WT tumors. We expect these studies will lead to an understanding
of the relevant determinants of immune response after PDT that could be rapidly applied to patient-selection
and improvement in outcome for PDT for cancer.
We have previously shown that a conjugate (MA-ce6) between maleylated serum albumin and the photosensitizer chlorin(e6) (ce6) is targeted in vitro to macrophages via class A scavenger receptors. We now report on the ability of this conjugate to localize in macrophage-rich atherosclerotic plaques in vivo. Both the conjugate and the free photosensitizer ce6 are studied after injection into New Zealand White rabbits that are rendered atherosclerotic by a combination of aortic endothelial injury and cholesterol feeding into normal rabbits. Rabbits are sacrificed at 6 and 24 h after injection and intravascular fluorescence spectroscopy is carried out by fiber-based fluorimetry in intact blood-filled arteries. Surface spectrofluorimetry of numbered excised aortic segments together with injured and normal iliac arteries is carried out, and quantified ce6 content by subsequent extraction and quantitative fluorescence determination of the arterial segments and also of nontarget organs. There is good agreement between the various techniques for quantifying ce6 localization, and high contrast between arteries from atherosclerotic and normal rabbits is obtained. Fluorescence correlates with the highest burden of plaque in the aorta and the injured iliac artery. The highest accumulation in plaques is obtained using MA-ce6 at 24 h. Free ce6 gives better accumulation at 6 h compared to 24 h. The liver, spleen, lung, and gall bladder have the highest uptake in nontarget organs. Macrophage-targeted photosensitizer conjugates may have applications in both detecting and treating inflamed vulnerable plaque.
Cancer is a leading cause of death among modern people largely due to metastatic disease. The ideal cancer treatment should target both the primary tumor and the metastases with minimal toxicity towards normal tissue. This is best accomplished by priming the body's immune system to recognize the tumor antigens so that after the primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the IV administration of photosensitizers followed by illumination of the tumor with red light producing reactive oxygen species leading to vascular shutdown and tumor cell death. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response, generation of tumor-specific antigens, and induction of heat-shock proteins. Combination regimens using PDT and immunostimulating treatments are likely to even further enhance post-PDT immunity. These immunostimulants are likely to include products derived from pathogenic microorganisms that are effectively recognized by Toll-like receptors and lead to upregulation of transcription factors for cytokines and inflammatory mediators. The following cascade of events causes activation of macrophages, dendritic and natural killer cells. Exogenous cytokine administration can be another way to increase PDT-induced immunity as well as treatment with a low dose of cyclophosphamide that selectively reduces T-regulatory cells. Although so far these combination therapies have only been used in animal models, their use in clinical trials should receive careful consideration.
One in 8 women in the United States will develop breast cancer during her lifetime and 40,000 die each year. Deaths are due to tumors that have metastasized despite local control. Photodynamic therapy (PDT) is a promising cancer treatment in which a photosensitizer (PS) accumulates in tumors and is subsequently activated by visible light of an appropriate wavelength. The energy of the light is transferred to molecular oxygen to produce reactive oxygen species that produce cell death and tumor ablation. Mechanisms include cytotoxicity to tumor cells, shutting down of the tumor vasculature, and the induction of a host immune response. The precise mechanisms involved in the PDT-mediated induction of anti-tumor immunity are not yet understood. Potential contributing factors are alterations in the tumor microenvironment via stimulation of proinflammatory cytokines and direct effects of PDT on the tumor that increase immunogenicity. We have studied PDT of 410.4 variant 4T1 tumors growing in the mammary fat pad (orthotopic) in Balb/c mice and which produce metastasis. We have shown that a PDT regimen that produces vascular shutdown and tumor necrosis leads to initial tumor ablation but the tumors recur at the periphery. We studied the combination of PDT with immunostimulating therapies. Low dose cyclophosphamide (CY) is a specific mechanism to deplete the regulatory T cells (CD4+CD25+), these cells play an important role in the immunosuppression activity of tumors. In combination with PDT that produces release of tumor specific antigens, this immunostimulation may lead to generation of cytotoxic CD8 T-lymphocytes that recognize and destroy the tumor. The second alternative therapy is the use of a novel combination of the immunostimulant CpG oligodeoxynucleotides (CpG-ODN) and PDT. CpG-ODN is recognized by Toll-like receptor 9 and directly or indirectly triggers B cells, NK cells, monocyte-macrophages and dendritic cells to proliferate, mature and secrete cytokines, chemokines and immunoglobulins. Both these novel combinations gave significantly enhanced therapeutic benefit not seen with single treatments alone. Tumors grew more slowly and mice lived significantly longer, although cures were rare. We propose that a rational choice of immune stimulant is an ideal addition to PDT regimens.
Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the <i>ex vivo</i> trial with fresh thick non-melanoma
skin cancer excisions and <i>in vivo</i> trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images
were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding <i>en face</i> frozen H&E sections. The
results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both <i>in vivo</i> and <i>in vitro</i>. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The
developed technique may provide an efficient real-time optical tool for detecting skin pathology.
Photodynamic therapy (PDT) is a modality for the treatment of cancer involving excitation of photosensitizers with harmless visible light producing reactive oxygen species. The major biological effects of PDT are apoptosis of tumor cells, destruction of the blood supply and activation of the immune system. The objective of this study is to compare in an animal model of metastatic cancer, PDT alone and PDT combined with low-dose cyclophosphamide (CY). Since the tumor we used is highly metastatic, it is necessary to generate anti-tumor immunity using PDT to both cure the primary tumor and prevent death from metastasis. This immunity may be potentiated by low dose CY. In our model we used J774 cells (a Balb/c reticulum cell sarcoma line with the characteristics of macrophages) and the following PDT regimen: benzoporphyrin derivative monoacid ring A (BPD, 2mg/kg injected IV followed after 15 min by 150 J/cm<sup>2</sup> of 690-nm light). CY (50 mg/kg i.p.) was injected 48 hours before light delivery. BPD-PDT led to complete regression of the primary tumor in more than half the mice but no permanent cures were obtained. BPD-PDT in combination with CY led to 60% permanent cures. CY alone gave no permanent cures but did provide a survival advantage. To probe permanent immunity cured animals were rechallenged with the same tumor cell line and the tumors were rejected in 71% of mice cured with BPD-PDT plus CY. We conclude that BPD-PDT in combination with CY gives best overall results and that this is attributable to immunological response activation in addition to PDT-mediated destruction of the tumor.
Cancer is a leading cause of death among modern people, largely due to metastatic disease. The ideal cancer treatment should destroy both the primary tumor and distant metastases with minimal toxicity to normal tissue. This is best accomplished by educating the body's immune system to recognize the tumor as foreign so that after the primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the IV administration of photosensitizers followed by illumination of the tumor with red light producing reactive oxygen species that eventually cause vascular shutdown and tumor cell apoptosis. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response, generation of tumor-specific antigens, and induction of heat-shock proteins. Combination regimens are likely to emerge in the future to even further enhance immunity. Green fluorescent protein is used as an optical reporter to non-invasively image the progression of mouse tumors, and in addition, may act as a foreign (jellyfish) antigen. We asked whether the response of tumor bearing mice to PDT differed when a non-immunogenic tumor cell line was transfected with GFP? We injected RIF-1 or RIF1-EGFP cells in the leg of C3H/HeN mice and both the cells and tumors grew equally well. We used two PDT protocols (benzoporphyrin derivative (BPD) with 15-minute interval or Photofrin with 24-hour interval). The results showed significant differences between the responses of RIF1 or RIF1-EGFP tumors after BPD or Photofrin PDT and complete cures and mouse survival when RIF-1 EGFP tumors were treated with BPD. This increased tumor response may be due to antibody-mediated cytotoxicity and the presence of an artificial tumor antigen (GFP) that can produce a CD8 T-cell response against the whole tumor. The presence of antibodies against EGFP in mouse serum correlates with the hypothesis.
Rupture of a vulnerable atherosclerotic plaque (VP) leading to coronary thrombosis is the chief cause of sudden cardiac death. VPs are angiographically insignificant lesions, which are excessively inflamed and characterized by dense macrophage infiltration, large necrotic lipid cores, thin fibrous caps, and paucity of smooth muscle cells. We have recently shown that chlorin(e6) conjugated with maleylated albumin can target macrophages with high selectivity via the scavenger receptor. We report the potential of this macrophage-targeted fluorescent probe to localize in VPs in a rabbit model of atherosclerosis, and allow detection and/or diagnosis by fluorescence spectroscopy or imaging. Atherosclerotic lesions were induced in New Zealand White rabbit aortas by balloon injury followed by administration of a high-fat diet. 24-hours after IV injection of the conjugate into atherosclerotic or normal rabbits, the animals were sacrificed, and aortas were removed, dissected and examined for fluorescence localization in plaques by fiber-based spectrofluorimetry and confocal microscopy. Dye uptake within the aortas was also quantified by fluorescence extraction of samples from aorta segments. Biodistribution of the dye was studied in many organs of the rabbits. Surface spectrofluorimetry after conjugate injection was able to distinguish between plaque and adjacent aorta, between atherosclerotic and normal aorta, and balloon-injured and normal iliac arteries with high significance. Discrete areas of high fluorescence (up to 20 times control were detected in the balloon-injured segments, presumably corresponding to macrophage-rich plaques. Confocal microscopy showed red ce6 fluorescence localized in plaques that showed abundant foam cells and macrophages by histology. Extraction data on aortic tissue corroborated the selectivity of the conjugate for plaques. These data support the strategy of employing macrophage-targeted fluorescent dyes to detect VP by intravascular spectrofluorimetry. It may also be possible to use macrophage-targeted PDT to therapeutically modify inflammatory cell-laden VPs leading to plaque stabilization and reduction of sudden cardiovascular death.