Invited Paper
Immunological responses induced by the combination of phototherapy and immunotherapy in the treatment of metastatic tumors Wei R. Chena*, Mark F. Naylor b, Robert E. Nordquistc, T. Kent Teagued, e, f, and Hong Liug a
Biomedical Engineering Program, Department of Engineering and Physics, College of Mathematics and Science, University of Central Oklahoma, Oklahoma 73034, USA b Dermatology Section, University of Oklahoma College of Medicine, 1507 E 19th Street, Tulsa, Oklahoma 74104, USA c Advanced Cancer Therapies L.L.C., 14 NE 48th Street, Oklahoma City, OK 73105, USA d Department of Surgery, University of Oklahoma College of Medicine, 4502 E 41st Street, Tulsa, Oklahoma 74135, USA e Department of Pharmaceutical Sciences, University of Oklahoma College of Pharmacy, Tulsa, OK 74135, USA f Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, Tulsa, OK 74107, USA g Center for Bioengineering and School of Electrical and Computer Engineering, University of Oklahoma, Norman, Oklahoma 73019, USA Abstract Combination therapy using laser photothermal interaction and immunological stimulation has demonstrated its ability to induce immunological responses. Glycated chitosan (GC), an immunological stimulant, and imiquimod, a new type of immune response modifier (IRM), when used in conjunction with laser phototherapy, have shown to have a great immunological stimulation function. Specifically, imiquimod can help release cytokines from immunocompetent cells, stimulate TH1 lymphocyte responses (CD8+ T-cells), and recruit additional dendritic cells. To study the effects of immunoadjuvnats in combination of laser photo-irradiation, we treated animal tumors with laser-ICG-GC combination and late-stage melanoma patients with laser-ICG-imiquimod combination. At designated times, tumors, blood, and spleens in both treated and untreated animals were colleted for analysis. The major immunological indicators, such as IL-6, IL-12, IFN-gamma, CD4, and CD8 were analyzed. The same immunological analysis was also performed for melanoma patients treated by the laser-imiquimod combination.
Keywords: 805-nm laser, imiquimod, B16 melanoma, glycated chitosan, indocyanine green, immunological responses, mammary tumors, cancer treatment *
Correspondence: Phone: (405) 974-5198; Fax: (405) 974-3812; Email:
[email protected] Biophotonics and Immune Responses III, edited by Wei R. Chen, Proc. of SPIE Vol. 6857, 685707, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.762132
Proc. of SPIE Vol. 6857 685707-1 2008 SPIE Digital Library -- Subscriber Archive Copy
1. Introduction Phototherapies for cancer treatment often cannot simply rely on acute tumor destruction from the deposited light energy. The secondary reaction – light induced immune responses often plays a major role in cancer treatment. A good example is the photodynamic therapy (PDT), which utilizes a photochemical reaction through the release of toxic single oxygen for direct tumor cell destruction. It could, on the one hand, suppress immune responses [1-3]. On the other hand, more often than not, if the treatment conditions are right, PDT could induce strong immune responses to enhance cancer treatment [4-7]. One component of PDT induced immune responses is through inflammatory reaction, which can initiate activation of the host antitumor immunity [8-11]. Another example of phototherapy for inducing immune responses is the laser immunotherapy, which combines selective laser photothermal interaction and immunological stimulation for the treatment of metastatic tumors [12-15]. During the past 10 years, laser immunotherapy has been advanced from a novel concept to pre-clinical studies [16-20] and to the clinical trials [21-23]. The curative effects of laser immunotherapy and its ability to induce host immune responses have been very promising. Laser immunotherapy, with its effective acute tumor cell destruction and prolonged, potent immunological stimulation, could become an alternative treatment modality for metastatic tumors. To further understand its mechanisms, we conducted additional immunological studies using laser immunotherapy in animal studies as well as in patient treatment. Here we present our pre-clinical studies on immunological responses using GC-based laser immunotherapy for the treatment of mammary tumors. We also present our preliminary clinical results using imiquimod-based laser immunotherapy in the treatment of late-stage melanoma patients.
2. Materials and Methods 2.1
Components of Laser Immunotherapy
This special method consists of three major components: an infrared laser, a light absorbing dye, and an immunological stimulant. The laser is the 805-nm DIOMED 25 diode laser (DIOMEDICS, The Woodlands, TX). Indocyanine green (ICG) (Akorn, Inc., Buffalo Grove, IL) was used as the laser-absorbing dye for both pre-clinical and clinical studies. Glycated chitosan (GC), a compound synthesized by our lab, was used as the immunoadjuvant in our animal experiments. For patient studies, imiquimod (Aldara™, imiquimod 5% cream, 3M Pharmaceuticals, St. Paul, Minnesota) was used as the immunoadjuvant. 2.2
Procedures of Laser Immunotherapy
The laser energy was delivered through an optical fiber with a diffusion lens to provide a uniform laser beam. The power density was 1 w/cm2. The irradiation duration was 10 minutes. ICG solution was injected into the target tumor before laser irradiation in a concentration of 0.25%. In animal studies, ICG was used in a 0.2 ml volume per tumor. In patient studies, the same concentration of ICG was used and the injected volume of ICG was determined by the size of the tumor (25% to 50% of the tumor volume). When the tumor was pigmented, ICG was usually not used because of the strong light absorption by the tumor without dye enhancement. The immunological stimulant for animal studies is the injectable glycated chitosan (1.0%) in 0.2 ml volume per tumor. Imiquimod was used topically twice daily on the target surface of the patient in conjunction with the laser treatment. 2.3
Animal Models
EMT6 mammary tumor model in female mice was used in is study. EMT6 tumor cells (105 cells per animal) were injected subcutaneously into the back of the BALB/c mice.
Proc. of SPIE Vol. 6857 685707-2
DMBA-4 transplantable, metastatic mammary tumor model in female Wistar Furth rats was used in the experiments. Approximately 10 5 viable tumor cells were implanted subcutaneously in one of the inguinal fat pads to induce the primary tumors, which become palpable about seven days after implantation. 2.4
Treatment of tumors in rats
When the tumors in mice and in rats grew to a size of 0.5 to 1 cm, they were treated with different components of laser immunotherapy, including laser irradiation only, ICG injection only, GC injection only, laser-GC treatment, and laser-ICG-GC treatment. Tumor-bearing rats were sacrificed at different time points after the treatment and the tumor samples and animal spleens were collected for analysis of immunological responses. 2.5
Treatment of melanoma patients
Imiquimod was applied topically on the target surface of the patient twice daily, including both the tumors and surrounding areas. A treatment cycle (see Figure 1) is a 6-week therapy for each treated local area. If residual tumors were observed after the cycle, the patient was treated with a new cycle.
Imiquimod BID with Occlusion
0
1
2
3
IR Laser
4
5
6
Weeks of Treatment
IR Laser
Figure 1. Treatment cycle of laser immunotherapy using a near-infrared laser and topical application of immunological stimulant.
IRLaser IRLaser
Imiquimod
Wee ks of Tr ea tm ent
A B
0
2
B C D
B B C C D
4
6
8
10
B B C C D
B C D
B C D
B C D
Figure 2. Sample collections from patients treated by laser immunotherapy at different time points of the treatment cycle. A: HLA typing; B: Cytokine Luminex assays; C: CD8 & IFN-g assays; D: Serum antibody Western blots.
2.6
Histological studies for animal experiments
In the study using rat tumors, at designated time points after laser treatment, the entire tumor from each rat was colleted and fixed in 10% formalin, paraffin embedded, and then sectioned and stained with
Proc. of SPIE Vol. 6857 685707-3
hemotoxylin and eosin. The samples were examined under an optical microscope. Multiple sections of each tumor were studied and photomicrographs representing different areas from several different animals were selected and analyzed. 2.7
Immunological assays
The mouse tumors and spleens were collected at different time points for cytokines studies, particularly IL-6 and IL-12. Sera from patients were collected before, during, and after the laser immunotherapy treatment. The time schedule for blood sample collections for different assays in the clinical studies is given in Figure 2.
3. Results In the immunological studies using the EMT6 mammary tumors in mice, the IL10 and IL12P40 were detected under treatment by different components of laser immunotherapy. The IL10 to IL12P40 ratios in tumor samples and spleens were obtained. Among different groups, the complete laser immunotherapy treatment, laser-ICG-GC, resulted in the lowest ratio, particularly in the spleen of the treated animals. Such results indicate a long-term immune response since spleen is the major organ responsible to produce the immune cells. In the histochemical studies using DMBA-4 mammary tumors in rats, the full treatment using laser-ICG-GC also showed the greatest immune responses. Shown in Figure 3 is the center of a rat tumor treated by laser immunotherapy. The thermal injury is observed characterized by the loss of the density in the tumor cytoplasma. Multiple lymphocytes are also observed in the tumor sites. The vacuolization is seen in the tumor cytoplasma. There are also lymphocytes and immature plasma cells in the field (right edge of the Figure 3(B)). There are much more lymphocytes in the tumor samples treated by laser-ICG-GC combination than that in the tumor treated by laser, ICG, GC, or a combination of any two components of laser immunotherapy (data not shown). In our preliminary study of immune responses in the treated melanoma patients, sera were collected from three patients (one stage III and two stage IV). In one stage IV patient, most of the immunological parameters, including interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-α (TNF−α), reached the highest level two weeks after the completion of the laser immunotherapy, while the CD4 remained low throughout the treatment. The second stage IV patient, on the other hand, showed increased level of CD4 after the laser immunotherapy. The stage III patient showed a significant increase in IL-6 level but a low IL-12 level throughout the treatment cycle. This patient showed a below normal level of CD4 and CD8 throughout the treatment.
4. Discussion Our pre-clinical studies definitely showed laser immunotherapy induced cellular immune responses, in addition to the clear thermal damage of the tumors. In comparison with the treatment results using single or double components of laser immunotherapy, the laser-ICG-GC combination caused the most lymphcyte infiltration, as shown by the results presented here (Section 3). It is believed that the effective photothermal damage to the tumor cells at a level that the cells were not completed denatured preserved the tumor antigens for the immune system to recognize. The use of immunoadjuvant directly stimulated the host immune system and combined with the tumor antigens to direct the immune system to fight the residual tumors and remote metastases. Laser-ICG-GC also apparently significantly increased the levels of IL10 to IL12P40 ratios in both treated tumors and in spleens, indicating a strong anti-tumor immune response.
Proc. of SPIE Vol. 6857 685707-4
The cytokines are crucial in the treatment of cancers. Interleukin-6 (IL-6) is a pro-inflammatory cytokine secreted by T cells and macrophages to stimulate immune responses to trauma, especially burns or other tissue damage leading to inflammation. It can cause the temperature increase. In some of the patients treated by laser immunotherapy, the increased level of IL-6 had been observed.
.
a
(A)
(B) Figure 3. DMBA-4 tumors in rats 24 hours after treated by laser immunotherapy. The center of the tumor are shown with different magnifications: 20X (A) and 40X (B). The thermal injury is apparent. Lymphocytes are observed in the tumor sites. Immature plasma cells were also observed in (B).
Interleukin-12 (IL-12) is naturally produced by macrophages and human B-lymphoblastoid cells in response to antigenic stimulation. It is a T-cell stimulating factor, help the production of Interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). It can also enhance cytotoxic activities of NK cells and CD8+T lymphocytes. An increased level of IL-12 was also observed in some of the treated patients.
Proc. of SPIE Vol. 6857 685707-5
Tumor necrosis factor-alpha (TNF-α) is a cytokine involved in systemic inflammation. It is mainly secreted by macrophages to regulate immune cells and regulate biological processes such as cell proliferation, differentiation and apoptosis. The laser treatment has shown to be able to increase the level of TNF-α in some of the patients. Data also showed that laser immunotherapy induced other cellular activities, such as CD4 and CD8. Our current studies with limited data samples in animal and clinical studies could not provide a clear picture of laser immunotherapy induced effective anti-tumor immune responses. However, they do point to the active participation of the immune system in the treatment. It is believed that the positive pre-clinical and clinical outcomes are direct results of the immunological activities. Our future study along this direction will further guide us to develop an effective treatment modality and also will provide us a better understanding of the mechanism of the laser immunotherapy.
Acknowledgement This research was supported in part by grants from the National Institute of Health (P20 RR016478 from the INBRE Program of the National Center for Research Resources).
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Lynch DH, Haddad S, Vernon KJ, Ott MJ, Straight RC, and Jolles CJ. Systemic immunosuppression induced by photodynamic therapy (PDT) is adoptively transferred by macrophages. Photochemistry and Photobiology, 49, 453-458, 1989. Musser DA and Fiel RJ. Cutaneous photosensitizing and immunosuppressive effects of a series of tumor localizing porphyrins. Photochemistry and Photobiology, 53, 119-123, 1991. Elmets CA, Bowen KD. Immunological suppression in mice treated with hematoporphyrin derivative photoradiation. Cancer Research, 46, 1608-1611, 1986. Yamamoto N, Hoober JK, Yamamoto N, and Yamamoto S. Tumoricidal capacities of macrophages photodynamically activated with hematoporphyrin derivative. Photochemistry and Photobiology, 56, 245-250, 1992. Korbelik M and Krosl G. Enhanced macrophage cytotoxicity against tumor cells treated with photodynamic therapy. Photochemistry and Photobiology, 60, 497-502, 1994. Krosl G, Korbelik M, Krosl J, and Dougherty GJ. Potentiation of photodynamic therapy-elicited antitumor response by localized treatment with granulocyte-macrophage colony-stimulating factor. Cancer Research, 56, 3281-3286, 1996. Canti G, Lattuada D, Nicolin A, Taroni P, Valentini G, and Cubeddu R. Antitumor immunity induced by photodynamic therapy with aluminum disulfonated phthalocyanines and laser light. Anticancer Drugs, 5, 443-447, 1994. Ryter SW, Gomer CJ. Nuclear factor kappa B binding activity in mouse L1210 cells following photofrin II-mediated photosensitization. Photochem Photobiol., 58, 753-756, 1993. Kick G, Messer G, Goetz A, Plewig G, and Kind P. Photodynamic therapy induces expression of interleukin 6 by activation of AP-1 but not NF-kappa B DNA binding. Cancer Res., 55, 2373-2379, 1995. Gollnick SO, Lee BY, Vaughan L, Owczarczak B, and Henderson BW. Activation of the IL-10 gene promoter following photodynamic therapy of murine keratinocytes. Photochem Photobiol., 73, 170-177, 2001. Korbelik M. Induction of tumor immunity by photodynamic therapy. J Clin Laser Med Surg., 14, 329-334, 1996. Chen WR, Adams RL, Carubelli R, and Nordquist RE. Laser-photosensitizer assisted immunotherapy: A novel modality in cancer treatment. Cancer Letters, 115, 25-30, 1997.
Proc. of SPIE Vol. 6857 685707-6
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Chen WR, Robinson K, Adams RL, Singhal A and Nordquist RE. Anti-tumor immune responses induced by the treatment of photodynamic immunotherapy. SPIE Vol., 3254, 27-34, 1998. Chen WR, Liu H, Lucroy MD and Nordquist RE. Anti-tumor immunity induced by local photothermal-immunoadjuvant therapy in a poorly immunogenic metastatic tumor model. SPIE Proceedings, Vol., 4961, 18-23, 2003. Chen WR, Bartels KE, Sun J, Liu H, Nordquist RE and Korbelik M. Dye-enhanced selective photothermal laser-tissue interaction and photodynamic therapy in combination with immunoadjuvant for cancer treatment. SPIE Proceedings, Vol., 5254, 52-60, 2003. Chen WR, Ritchey JW, Bartels KE, Liu H, and Nordquist RE. Effect of different components of laser immunotherapy in treatment of metastatic tumors in rats. Cancer Research, 62, 4295-4299, 2002. Chen WR, Korbelik M, Sun J, Liu H, and Nordquist RE. Enhancement of glycated chitosan in laser cancer treatment. SPIE Proceedings, Vol., 5319, 80-86, 2004. Chen WR, Singhal AK, Liu H, and Nordquist RE. Laser immunotherapy induced antitumor immunity and its adoptive transfer. Cancer Research, 61, 459-461, 2001. Chen WR, Zhu W, Dynlacht JR, Liu H and Nordquist RE. Long-term tumor resistance induced by laser photo-immunotherapy. International Journal of Cancer, 81, 808-812, 1999. Chen WR, Mohamed A, Naylor MF, Bartels KE, Ritchey JW, Liu H, and Nordquist RE. Cellular immunological effects of laser irradiation and immunoadjuvant application. SPIE Proceedings, Vol., 6438, 64380B(1-11), 2007. Naylor MF, Nordquist RE, Teague TK, Perry LA, and Chen WR. In Situ Photoimmunotherapy for Melanoma: Preliminary Clinical Result. SPIE Proceedings, Vol., 6087, 608709-1 – 608709-7, 2006. Naylor MF, Chen WR, Teague TK, Perry LA, and Nordquist RE. In Situ Photo Immunotherapy: A Tumor-Directed Treatment Modality for Melanoma. British Journal of Dermatology, 155, 12871292, 2006. Naylor MF, Nordquist RE, Teague TK, Perry LA, and Chen WR. In Situ Photoimmunotherapy for Melanoma: An Ongoing Phase I Clinical Trial. SPIE Proceedings, Vol., 6438, 643807(1-9), 2007.
Proc. of SPIE Vol. 6857 685707-7
