Cancer photo-immunotherapy: from bench to bedside.

Targeted therapy and immunotherapy in combination is considered the ideal strategy for treating metastatic cancer, as it can eliminate the primary tumors and induce host immunity to control distant metastases. Phototherapy, a promising targeted therapy, eradicates primary tumors using an appropriate dosage of focal light irradiation, while initiating antitumor immune responses through induced immunogenic tumor cell death. Recently, phototherapy has been employed to improve the efficacy of immunotherapies such as chimeric antigen receptor T-cell therapy and immune checkpoint inhibitors. Phototherapy and immunoadjuvant therapy have been used in combination clinically, wherein the induced immunogenic cell death and enhanced antigen presentation synergy, inducing a systemic antitumor immune response to control residual tumor cells at the treatment site and distant metastases. This review summarizes studies on photo-immunotherapy, the combination of phototherapy and immunotherapy, especially focusing on the development and progress of this unique combination from a benchtop project to a promising clinical therapy for metastatic cancer.

Phototherapy using immunologically modified carbon nanotubes to potentiate checkpoint blockade for metastatic breast cancer.

Metastasis is the major cause of cancer-death. Checkpoint inhibition shows great promise as an immunotherapeutic treatment for cancer patients. However, most currently available checkpoint inhibitors have low response rates. To augment the antitumor efficacy of checkpoint inhibitors, such as CTLA-4 antibodies, a single-walled carbon nanotube (SWNT) modified by a novel immunoadjuvant, glycated chitosan (GC), was used for the treatment of metastatic mammary tumors in mice. We treated the primary tumors by intratumoral administration of SWNT-GC, followed with irradiation with a 1064-nm laser to achieve local ablation through photothermal therapy (PTT). The treatment induced a systemic antitumor immunity which inhibited lung metastasis and prolonged the animal survival time of treated. Combining SWNT-GC-laser treatment with anti-CTLA-4 produced synergistic immunomodulatory effects and further extended the survival time of the treated mice. The results showed that the special combination, PTT + SWNT-GC + anti-CTLA, could effectively suppress primary tumors and inhibit metastases, providing a new treatment strategy for metastatic cancers.

Nanotechnology-based photoimmunological therapies for cancer.

Phototherapy is a non-invasive or minimally invasive therapeutic strategy. Immunotherapy uses different immunological approaches, such as antibodies, vaccines, immunoadjuvants, and cytokines to stimulate the host immune system to fight against diseases. In cancer treatment, phototherapy not only destroys tumor cells, but also induces immunogenic tumor cell death to initiate a systemic anti-tumor immune response. When combined with immunotherapy, the effectiveness of phototherapy can be enhanced. Because of their special physical, chemical, and sometimes immunological properties, nanomaterials have also been used to enhance phototherapy. In this article, we review the recent progress in nanotechnology-based phototherapy, including nano-photothermal therapy, nano-photochemical therapy, and nano-photoimmunological therapy in cancer treatment. Specifically, we focus on the immunological responses induced by nano-phototherapies.

Treatment of advanced melanoma with laser immunotherapy and ipilimumab.

Immunotherapy has become a promising modality for melanoma, especially using checkpoint inhibitors, which revive suppressed T cells against the cancer. Such inhibitors should work better when combined with other treatments which could increase the number and quality of anti-tumor T cells. We treated one patient with advanced (stage IV) melanoma, using the combination of laser immunotherapy (LIT), a novel immunological approach for metastatic cancers that has been shown to stimulate adaptive immunity, and ipilimumab. The patient was treated with LIT, followed with one course of ipilimumab 3 months after the beginning of LIT. After LIT treatment, all treated cutaneous melanoma in head and neck cleared completely. After the application of ipilimumab, all the tumor nodules in the lungs decreased. The patient had remained tumor free for one year. While anecdotal, the responses seen in this patient support the hypothesis that laser immunotherapy increases the number and quality of anti-tumor T cells so that ipilimumab and other checkpoint inhibitors are more effective in enhancing the therapeutic effects. Picture: Schematic of treatment using laser immunotherapy and ipilimumab on a stage IV melanoma patient.

Advances in strategies and methodologies in cancer immunotherapy.

Since the invention of Coley's toxin by William Coley in early 1900s, the path for cancer immunotherapy has been a convoluted one. Although still not considered standard of care, with the FDA approval of trastuzumab, Provenge and ipilimumab, the medical and scientific community has started to embrace the possibility that immunotherapy could be a new hope for cancer patients with otherwise untreatable metastatic diseases. This review aims to summarize the development of some major strategies in cancer immunotherapy, from the earliest peptide vaccine and transfer of tumor specific antibodies/T cells to the more recent dendritic cell (DC) vaccines, whole cell tumor vaccines, and checkpoint blockade therapy. Discussion of some major milestones and obstacles in the shaping of the field and the future perspectives is included. Photoimmunotherapy is also reviewed as an example of emerging new therapies combining phototherapy and immunotherapy.

InCVAX--a novel strategy for treatment of late-stage, metastatic cancers through photoimmunotherapy induced tumor-specific immunity.

A novel, promising potential cancer vaccine strategy was proposed to use a two-injection procedure for solid tumors to prompt the immune system to identify and systemically eliminate primary and metastatic cancers. The two-injection procedure consists of local photothermal application on a selected tumor intended to liberate whole cell tumor antigens, followed by a local injection of an immunoadjuvant that consists of a semi-synthetic functionalized glucosamine polymer, N-dihydro-galacto-chitosan (GC), which is intended to activate antigen presenting cells and facilitate an increased uptake of tumor antigens. This strategy is thus proposed as an in situ autologous cancer vaccine (inCVAX) that may activate antigen presenting cells and expose them to tumor antigens in situ, with the intention of inducing a systemic tumor specific T-cell response. Here, the development of inCVAX for the treatment of metastatic cancers in the past decades is systematically reviewed. The antitumor immune responses of local photothermal treatment and immunological stimulation with GC are also discussed. This treatment approach is also commonly referred to as laser immunotherapy (LIT).

Chitin, chitosan, and glycated chitosan regulate immune responses: the novel adjuvants for cancer vaccine.

With the development of cancer immunotherapy, cancer vaccine has become a novel modality for cancer treatment, and the important role of adjuvant has been realized recently. Chitin, chitosan, and their derivatives have shown their advantages as adjuvants for cancer vaccine. In this paper, the adjuvant properties of chitin and chitosan were discussed, and some detailed information about glycated chitosan and chitosan nanoparticles was also presented to illustrate the trend for future development.

Assessment of thermal effects of interstitial laser phototherapy on mammary tumors using proton resonance frequency method.

Laser immunotherapy (LIT) uses a synergistic approach to treat cancer systemically through local laser irradiation and immunological stimulation. Currently, LIT utilizes dye-assisted noninvasive laser irradiation to achieve selective photothermal interaction. However, LIT faces difficulties treating deeper tumors or tumors with heavily pigmented overlying skin. To circumvent these barriers, we use interstitial laser irradiation to induce the desired photothermal effects. The purpose of this study is to analyze the thermal effects of interstitial irradiation using proton resonance frequency (PRF). An 805-nm near-infrared laser with an interstitial cylindrical diffuser was used to treat rat mammary tumors. Different power settings (1.0, 1.25, and 1.5 W) were applied with an irradiation duration of 10 min. The temperature distributions of the treated tumors were measured by a 7 T magnetic resonance imager using PRF. We found that temperature distributions in tissue depended on both laser power and time settings, and that variance in tissue composition has a major influence in temperature elevation. The temperature elevations measured during interstitial laser irradiation by PRF and thermocouple were consistent, with some variations due to tissue composition and the positioning of the thermocouple's needle probes. Our results indicated that, for a tissue irradiation of 10 min, the elevation of rat tumor temperature ranged from 8 to 11°C for 1 W and 8 to 15°C for 1.5 W. This is the first time a 7 T magnetic resonance imager has been used to monitor interstitial laser irradiation via PRF. Our work provides a basic understanding of the photothermal interaction needed to control the thermal damage inside a tumor using interstitial laser treatment. Our work may lead to an optimal protocol for future cancer treatment using interstitial phototherapy in conjunction with immunotherapy.

Preliminary safety and efficacy results of laser immunotherapy for the treatment of metastatic breast cancer patients.

We report our preliminary results of a pilot clinical trial of late-stage breast cancer patients treated by laser immunotherapy (LIT), a local intervention using an 805 nm laser for non-invasive irradiation, indocyanine green for selective thermal effect, and immunoadjuvant (glycated chitosan) for immunological stimulation. Ten breast cancer patients were enrolled in this study; all patients were considered to be out of other available treatment options. Toxicity was individually evaluated through physical exams and laboratory tests. Adverse reactions only occurred in the area of treatment due to photothermal injury and local administration of immunoadjuvant. No grade 3 or 4 side effects were observed. Treatment efficacy of LIT was also evaluated by physical examination and tomography. In 8 patients available for evaluation, the objective response rate was 62.5% and the clinical beneficial response rate was 75%. While the study is still ongoing, the initial outcomes of this clinical trial show that LIT is well tolerated and is promising in the treatment of metastatic breast cancer.

Long-term effect on EMT6 tumors in mice induced by combination of laser immunotherapy and surgery.

Laser immunotherapy (LI) has been demonstrated to be a promising modality for cancer treatment. The present study was designed to further investigate the impact of LI combined with surgery. LI consists of a near-infrared laser, a light-absorbing dye (indocyanine green, ICG), and an immunostimulant (glycated chitosan, GC). ICG and GC were intratumorally injected, followed by laser irradiation. Female BALB/c mice bearing EMT6 tumor cells were divided into 4 groups: control, LI, LI followed by immediate surgery resection of residual tumor (LI + S(0wk)), and LI followed by surgical removal of residual tumor after 1 week (LI + S(1wk)). Successfully treated mice from all treatment groups were rechallenged twice with 10(5) and 5 × 10(5) EMT6 cells, respectively. The LI + S(1wk) group had the highest survival rate (72%) after 90 days, whereas the mice survival rates of the LI + S(0wk), LI, and control groups were 50%, 46%, 0%, respectively. The median survival times of control, LI, LI + S(0wk), and LI + S(1wk) groups were 32, 66, 74, and 90 days, respectively. Survival rates of the treated mice after the first and second tumor rechallenges, ranging from 73% to 95%, were not significantly different among the 4 groups (P > .05). The results show that LI is a useful tool for the treatment of tumor-bearing mice. Long-term antitumor effect can be induced by LI. They also indicate that combination of LI with surgery can further improve the therapeutic efficiency of LI.

Clinical effects of in situ photoimmunotherapy on late-stage melanoma patients: a preliminary study.

Metastatic melanoma is a skin cancer with poor prognosis. In situ photoimmunotherapy (ISPI) is a promising modality for the treatment of metastatic melanoma that combines local, selective photothermal therapy with immunological stimulation. A preliminary clinical study was conducted to evaluate the safety and therapeutic effects of ISPI for late-stage melanoma patients using imiquimod as the immune modifier. Eleven patients received ISPI in one or multiple 6-week treatment cycles applied to a 200-cm2 treatment site, which usually contained multiple cutaneous metastases. ISPI consisted of three main components applied directly to the cutaneous metastases: 1) local application of topical imiquimod; 2) injection of indocyanine green (ICG); and 3) an 805 nm laser for local irradiation. All patients completed at least one cycle of treatment. The most common adverse effects were rash and pruritus at the treatment sites. No grade 4 toxicity was observed. Complete response was observed in six patients. All lesions in the treatment area of the patients responded to ISPI, eight of which achieved complete local response (CLR). CLR was observed in the non-treatment site (regional) lesions in four patients. Five patients were still alive at the time of last follow-up. The probability of 12-month overall survival was 70%. This study demonstrates that ISPI with imiquimod is safe and well tolerated. The patient response rate is promising. ISPI can be easily applied on an outpatient basis and can be combined with other modalities to improve the therapeutic response of metastatic melanoma.

Glycated chitosan as a new non-toxic immunological stimulant.

Chitosan is capable of stimulating immune responses. However, because chitosan is not water soluble, it has limited biological applications. By attaching galactose molecules to the chitosan molecules, a new water-soluble compound, glycated chitosan (GC), was synthesized. GC was designed for immune stimulations in combination with phototherapies in the treatment of metastatic tumors. To investigate the possible toxicity of GC, cultures of normal and tumor cells were incubated with GC of different concentrations and the cell viabilities were determined. For in vivo studies, GC solution was fed or injected to animals and its toxicity was determined through observations of animals and histological examinations of vital organs. No toxic effects of GC were observed in cultured cells or in animal studies. In addition, the immunological effect of GC was investigated through its stimulation of TNFalpha secretion by macrophages in vitro. In vivo studies showed enhancement of the survival of laser immunotherapy-treated rats bearing metastatic mammary tumors. Our in vitro and in vivo results indicated that GC was a strong immunological stimulant. Its non-toxic nature and immunological activity make GC a potential immunoadjuvant for treatment of metastatic tumors.

Photoimmunotherapy for cancer treatment.

Phototherapy, with its effective dose control and light delivery, has become a promising modality for treating malignant and nonmalignant diseases. Photochemical interaction, specifically photodynamic therapy (PDT), and photothermal interaction have been the primary mechanisms for direct cell destruction in the treatment of cancers. Preclinical studies demonstrate that, in addition to direct local cytotoxicity, PDT can also induce systemic immune responses, which may enhance therapeutic effects on primary tumors and on metastases at distant sites. Selective photothermal therapy, using an in situ application of light-absorbing dye, has also proven to be an effective method for local treatment of tumors. When combined with immunotherapy, the effects of phototherapy can be amplified, potentially making the photoimmunotherapy a systemic treatment modality. This phototherapy-immunotherapy combination, particularly in conjunction with immunoadjuvant, has been used in preclinical studies. The efficacy and long-term effects of such a combination are summarized and the recent experimental results are presented. A new immunoadjuvant, glycated chitosan (GC), has been used to enhance photochemical and photothermal therapies. The PDT-GC combination in the treatment of mammary tumors and lung tumors in mice provided significant improvement in the long-term survival of tumor-bearing animals. The use of GC in dye-assisted laser photothermal therapy also provided long-term curative effects and antitumor immune responses in the treatment of metastatic tumors in rats. The immune responses induced by phototherapy and enhanced by immunotherapy could become important mechanism in the control of metastatic tumors.

Enhancement of laser cancer treatment by a chitosan-derived immunoadjuvant.

A chitosan derivative, glycated chitosan (GC), has been used as an immunostimulant for cancer treatment in laser immunotherapy. The function of GC is to enhance the host immune response after direct cancer cell destruction by a selective laser photothermal interaction. To further test its effects, laser immunotherapy was extended to include several different adjuvants for immunological stimulation and to include photodynamic therapy (PDT) as a different tumor-destruction mechanism. Complete Freund (CF) adjuvant, incomplete Freund (IF) adjuvant and Corynebacterium parvum (CP) were selected for treatment of metastatic mammary tumors in rats, in combination with a selective photothermal interaction. The solution of the immunoadjuvants admixed with indocyanine green (ICG), a light-absorbing dye, was injected directly into the tumors, followed by noninvasive irradiation of an 805 nm laser. Combined with PDT, in the treatment of tumors in mice, GC was administered peritumorally immediately after laser irradiation. The survivals of treated animals were compared with untreated control animals. In the treatment of rat tumors, CF, IF and CP raised the cure rates from 0% to 18%, 7% and 9%, respectively. In comparison, GC resulted in a 29% long-term survival. In the treatment of EMT6 mammary sarcoma in mice, GC of 0.5% and 1.5% concentrations increased the cure rates of Photofrin-based PDT treatment from 38% to 63% and 75%, respectively. In the treatment of Line 1 lung adenocarcinoma in mice, a 1.67% GC solution enabled a noncurative meso-substituted tetra(meta-hydroxy-phenyl)chlorin-based PDT to cure 37% of the tumor-bearing mice. The experimental results of this study confirmed our previous studies, showing that immunoadjuvants played an active role in laser-related cancer treatment and that GC significantly enhanced the efficacy of laser cancer treatment.

Induced antitumor immunity against DMBA-4 metastatic mammary tumors in rats using laser immunotherapy.

Induced antitumor immunity is a highly effective and long-term cure for cancer, particularly for metastatic tumors. Laser immunotherapy was developed to induce such an immunologic response. It involves intratumoral administration of a light-absorbing dye and a specially formulated immunoadjuvant, followed by noninvasive irradiation of a near-infrared laser. Treatment of DMBA-4 metastatic mammary tumors in rats with this approach has resulted in local control of primary tumors and eradication of untreated distant metastases. After laser immunotherapy, rats were resistant to tumor rechallenge and developed immunity, which could be adoptively transferred. To better understand the immunity induced in this tumor model, immunization using freeze-thaw DMBA-4 cell lysates was performed, followed by tumor challenge 21 days later. Tumor cell lysate immunization delayed the emergence of metastases but did not provide immunity against the tumor challenge. Also performed was surgical resection of primary tumors before the observation of metastatic tumors. Removal of primary tumors was unsuccessful at changing the course of tumor progression. Tumors re-emerged at the primary sites, and metastases developed at multiple remote sites. In contrast, tumor-bearing rats successfully treated by laser immunotherapy experienced tumor regression and eradication and developed strong resistance to repeated challenges by tumor cells of the same type. Our results show that laser immunotherapy could have potential for the treatment of metastatic tumors by inducing tumor-specific, long-lasting immunity.

A phase I human trial of mitoguazone and gemcitabine sequential bi-weekly treatment of cancer patients.

Our previous studies have demonstrated the existence of synergism in a combination therapy using mitoguazone and gemcitabine when the mitoguazone is administered 24 hours before gemcitabine. Based on the cell culture and animal experimental results, a phase I clinical trial was performed in order to determine the toxicity of the combined treatment. Mitoguazone and gemcitabine were administered sequentially: mitoguazone on day 1 and gemcitabine on day 2. This cycle was repeated every 2 weeks. The dosages of these two drugs were varied between patients. Ten patients were enrolled in the study. Six patients began treatment at dose level 1 (mitoguazone 500 mg/m2, gemcitabine 1500 mg/m2), three at dose level 2 (mitoguazone 500 mg/m2, gemcitabine 2000 mg/m2), and one at dose level 3 (mitoguazone 600 mg/m2, gemcitabine 2000 mg/m2). Dose-limiting toxicity (DLT) was only observed in two patients treated at dose level 1 and one patient treated at dose level 3, while all the other patients only experienced nonhematologic toxicity, such as asthenia and mucositis. Two melanoma patients showed responses (one partial and one minor) to the treatment. One lymphoma patient also showed a brief partial response. This phase I trial indicated that the combination of mitoguazone and gemcitabine had limited but noticeable activity for treatment of cancer patients. Further study on the toxicity and on the effect of the scheduled mitoguazone-gemcitabine combination is needed.

Detection of anti-tumor immunity induced by laser immunotherapy.

Metastatic mammary tumors in rats were treated by laser immunotherapy. Tumors injected by a laserabsorbing dye and an immunoadjuvant were irradiated non-invasively by a near-infrared laser. The successfully cured rats developed a long-term resistance to repeated tumor challenges. Using the sera from cured rats as the source of primary antibodies, immune responses induced by laser immunotherapy were observed in both cellular and molecular levels using histochemical assays and Western blot analysis.

Laser immunotherapy: a novel treatment modality for metastatic tumors.

Laser immunotherapy is a novel approach for the treatment of metastatic tumors. It combines a selective photothermal laser-tissue interaction for direct tumor destruction and an immunoadjuvant-directed simulation for immune responses. In experiments using a rat metastatic tumor model, laser immunotherapy resulted in the eradication of both treated primary tumors and untreated metastases at remote sites. It also induced anti-tumor resistance.

Synergistic effect of sequential administration of mitoguazone (MGBG) and gemcitabine in treating tissue cultured human breast cancer cells and mammary rat tumors.

Modulation of cancer chemotherapeutic drugs has been attempted to increase efficacy and overcome resistance to the chemotherapeutic agent. Studies have shown schedule-dependent interactions in combined use of chemotherapeutic drugs. Mitoguazone (MGBG), an old drug with possible modulating activity, was used in combination with gemcitabine, a relatively new cancer drug, in treating tissue cultured human breast cancer cells and mammary rat tumors. Tissue cultured BOT-2 cancer cells were first treated with varying concentrations of gemcitabine and MGBG, independently. Combinations of the two drugs were then used with different scheduled administrations. Marked synergistic activity was found between gemcitabine and MGBG when the MGBG was given first, followed by gemcitabine 24 hours later. A non-toxic dose of MGBG enhanced the toxicity of gemcitabine by eight orders of magnitude using MTT assays in the tissue cultured human breast cancer cell study. The sequential administration of MGBG and gemcitabine also increased the survival rate of rats bearing mammary tumors in our pilot animal study.

Effect of different components of laser immunotherapy in treatment of metastatic tumors in rats.

Induction of a long-term tumor-specific immunity is the ultimate cure of metastatic cancers. Laser immunotherapy is a novel approach that aims at the tumor-directed stimulation of the immune system of the host. It involves an intratumor administration of a laser-absorbing dye and an immunoadjuvant, followed by noninvasive laser irradiation. Previous studies using glycated chitosan (GC) as immunoadjuvant and indocyanine green (ICG) as laser-absorbing dye have shown positive effects of the treatment on metastatic breast tumors in rats. In vivo experiments showed promising results such as: (a) eradication of treated primary tumors; (b) regression of untreated metastases; (c) induced antitumor immune response; and (d) long-term resistance to tumor rechallenge. In this study, rats bearing metastatic breast tumors and metastatic prostate tumors were treated with various combinations of the three components of laser immunotherapy. The rat survival rates and profiles of primary and metastatic tumors, after treatment by individual components and various combinations of the components, were analyzed. In the treatment of breast tumors, all of the experimental groups without immunoadjuvant showed little or no positive effect. The use of GC, either by itself or in combination with other components, had a noticeable impact on the survival rate of tumor-bearing rats. However, it was the combination of all of the three components that resulted in the highest cure rate. Three different concentrations of GC, 0.5, 1, and 2%, were also used to treat the metastatic breast tumors. The results showed that 1% GC was most effective in laser immunotherapy. In the treatment of metastatic prostate tumors, both the laser-ICG and laser-ICG-GC treatments significantly reduced the growth of primary tumors and lung metastases. Long-term survival of the rats bearing the prostate tumors was also observed after the laser immunotherapy treatment in our preliminary studies. These results revealed the important function of the immunoadjuvant in laser immunotherapy.