Host Immunity Following Near-Infrared Photoimmunotherapy Is Enhanced with PD-1 Checkpoint Blockade to Eradicate Established Antigenic Tumors.

Near-infrared photoimmunotherapy (NIR-PIT) induces immunogenic cell death but has mostly failed to induce durable antitumor responses in syngenic tumor mouse models. We hypothesized that adaptive immune resistance could be limiting durable responses after treatmemt with NIR-PIT. We investigated the effects of combining NIR-PIT targeting cell-surface CD44 and PD-1 blockade in multiple syngeneic tumor models. In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade reversed adaptive immune resistance, resulting in both enhanced preexisting tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses correlated with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT, as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic antitumor immunity. Mice that cleared tumors resisted subsequent tumor challenge, indicating the presence of systemic immune memory. Cumulatively, these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in antigenic syngeneic models of cancer.

Activatable Near-Infrared Fluorescence Imaging Using PEGylated Bacteriochlorin-Based Chlorin and BODIPY-Dyads as Probes for Detecting Cancer.

Near infrared (NIR) fluorescent probes are attractive tools for biomedical in vivo imaging due to the relatively deeper tissue penetration and lower background autofluorescence. Activatable probes are turned on only after binding to their target, further improving target to background ratios. However, the number of available activatable NIR probes is limited. In this study, we introduce two types of activatable NIR fluorophores derived from bacteriochlorin: chlorin-bacteriochlorin energy-transfer dyads and boron-dipyrromethene (BODIPY)-bacteriochlorin energy-transfer dyads. These fluorophores are characterized by multiple narrow excitation bands with relatively strong emission in the NIR. Targeted bacteriochlorin-based antibody or peptide probes have been previously limited by aggregation after conjugation. Polyethylene glycol (PEG) chains were added to improve the hydrophilicity without altering pharmacokinetics of the targeting moieties. These PEGylated bacteriochlorin-based activatable fluorophores have potential as targeted activatable, multicolor NIR fluorescent probes for in vivo applications.

Near infrared photoimmunotherapy using a fiber optic diffuser for treating peritoneal gastric cancer dissemination.

BACKGROUND: Peritoneal dissemination (PD) of abdominal malignancies is a common form of metastasis and its presence signals a poor prognosis. New treatment is required for patients with PD. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate (APC). In this study, we investigate in vitro and in vivo efficacy of trastuzumab (tra)-IR700DX NIR-PIT on a human epidermal growth factor receptor type 2 (HER2)-positive gastric cancer cell line. METHODS: NIR-PIT effects were investigated in vitro and in vivo. Disseminated peritoneal implants mice were separated into 5 groups: (1) no treatment; (2) tra-IR700 i.v. only; (3) NIR light only; (4) NIR-PIT; (5) repeated NIR-PIT. The peritoneal cavity was irradiated with NIR light using a fiber optic diffuser delivered through the catheter. RESULTS: Specific binding and cell-specific killing was observed after NIR-PIT in vitro. In the in vivo study, fluorescence endoscopy showed high tumor accumulation of tra-IR700 within tumors. Significantly prolonged survival was achieved in the three treatment groups (tra-IR700 i.v. only, NIR-PIT, and repeated NIR-PIT groups) compared with control and NIR light only group (p < 0.05 for tra-IR700 i.v. only, p < 0.01 for NIR-PIT, and p < 0.0001 for repeated NIR-PIT). Moreover, most prolonged survival was shown for the repeated NIR-PIT group (p < 0.0001 vs tra-IR700 i.v. only, p < 0.01 vs NIR-PIT). CONCLUSION: NIR-PIT using a fiber optic diffuser to deliver light is a promising candidate for the treatment of disseminated peritoneal metastases and could be readily translated to humans.

Pitfalls on sample preparation for ex vivo imaging of resected cancer tissue using enzyme-activatable fluorescent probes.

In vivo and ex vivo fluorescence imaging-assisted surgery can aid in determining the margins of tumors during surgical resection. While a variety of fluorescent probes have been proposed for this task, small molecule enzyme-activatable fluorescent probes are ideal for this application. They are quickly activated at tumor sites and result in bright signal with little background, resulting in high sensitivity. Testing in resected specimens, however, can be difficult. Enzymes are usually stable after freezing and thawing but catalytic reactions are generally temperature-dependent. Therefore, tissue sample temperature should be carefully considered. In this study two enzyme activatable probes, γ-glutamylhydroxymethyl rhodamine green (gGlu-HMRG) that reacted with γ-glutamyltransferase and SPiDER-ßGal that reacted with ß-galactosidase, were employed to determine the effects of temperature on fluorescence signal kinetics in both fresh and frozen and then thawed ex vivo experimental ovarian cancer tissue samples. The results suggest γ-glutamyltransferase was less sensitive to temperature than ß-galactosidase. Fresh samples showed higher fluorescence signals of gGlu-HMRG compared with thawed samples likely because the freeze-thaw cycle decreased the rate of internalization of the activated probe into the lysosome. In contrast, no significant difference of SPiDER-ßGal fluorescence signal was observed between fresh and frozen tissues. In conclusion, although imaging of fresh samples at 37°C is the best condition for both probes, successful imaging with gGlu-HMRG could be achieved even at room temperature with thawed samples. We demonstrate that temperature regulation and tissue handling of resected tissue are two pitfalls that may influence ex vivo imaging signals with enzyme-activatable fluorescent probes.

Near infrared photoimmunotherapy targeting bladder cancer with a canine anti-epidermal growth factor receptor (EGFR) antibody.

Anti-epidermal growth factor receptor (EGFR) antibody therapy is used in EGFR expressing cancers including lung, colon, head and neck, and bladder cancers, however results have been modest. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate which is activated by NIR light. NIR-PIT is in clinical trials in patients with recurrent head and neck cancers using cetuximab-IR700 as the conjugate. However, its use has otherwise been restricted to mouse models. This is an effort to explore larger animal models with NIR-PIT. We describe the use of a recombinant canine anti-EGFR monoclonal antibody (mAb), can225IgG, conjugated to the photo-absorber, IR700DX, in three EGFR expressing canine transitional cell carcinoma (TCC) cell lines as a prelude to possible canine clinical studies. Can225-IR700 conjugate showed specific binding and cell-specific killing after NIR-PIT on EGFR expressing cells in vitro. In the in vivo study, can225-IR700 conjugate demonstrated accumulation of the fluorescent conjugate with high tumor-to-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 µg of can225-IR700 i.v. only; (3) NIR light exposure only; (4) 100 µg of can225-IR700 i.v., NIR light exposure. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and significantly prolonged survival was achieved (p < 0.001 vs. other groups) in the treatment groups. In conclusion, NIR-PIT with can225-IR700 is a promising treatment for canine EGFR-expressing cancers, including invasive transitional cell carcinoma in pet dogs, that could provide a pathway to translation to humans.

Endoscopic near infrared photoimmunotherapy using a fiber optic diffuser for peritoneal dissemination of gastric cancer.

Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate (APC) which is activated by near infrared light. Here, we describe the efficacy of endoscopic NIR-PIT using the APC trastuzumab-IR700DX (tra-IR700) in the setting of human epidermal growth factor 2 positive (HER2 + ) gastric carcinoma with peritoneal disseminations. In this in vivo study, fluorescence endoscopy showed high tumor accumulation of tra-IR700 within disseminated peritoneal implants. Mice with disseminated peritoneal gastric cancer were separated into 4 groups: (i) control (no treatment); (ii) tra-IR700 i.v. only; (iii) NIR light only; and (iv) endoscopic NIR-PIT. NIR light irradiation was carried out through a fiber optic diffuser under endoscopic guidance. In vivo bioluminescence images showed significantly greater therapeutic effect in the endoscopic NIR-PIT group than that in the control groups (P < .01 vs other control groups). Histological analysis showed diffuse cancer cell death in NIR-PIT-treated tumors. In conclusion, NIR-PIT with NIR light delivered via an endoscopic fiber optic diffuser is a promising method for the treatment of peritoneal dissemination of gastric cancer. Moreover, this technique could be readily used in other types of cancers with peritoneal dissemination provided that suitable antibodies could be found.

Interstitial near-infrared photoimmunotherapy: effective treatment areas and light doses needed for use with fiber optic diffusers.

Near-infrared photoimmunotherapy (NIR-PIT), a promising cancer therapy utilizing an antibody-photoabsorber conjugate (APC) and NIR light, which induces rapid necrotic cell death only in APC-bound cells. Effective NIR-PIT in mouse models has been achieved using superficial light illumination (SLI) with light emitting diodes (LEDs) or lasers, but in the clinical setting, fiber optic diffusers have been employed to deliver light to deeper tumors. However, the performance of NIR light in tissue delivered by fiber optic diffusers is poorly understood. Here, we investigated NIR-PIT using a cylindrical fiber optic diffuser in a mouse model of A431 tumors. NIR-PIT with 100 J/cm, the same light dose used in clinical trials of NIR-PIT, was applied after insertion of the diffuser within the tumor bed, and then both bioluminescence and fluorescence imaging were analyzed to assess the therapeutic efficacy. The diffuser can deliver adequate NIR light dose for effective NIR-PIT to the A431 tumor at a distance of approximately 1 cm around the light source at 100 J/cm. At 50 J/cm NIR light effective NIR-PIT was reduced to a distance of 5 - 7 mm diameter around the light source. These results indicate that the energy of interstitial light (measured in Joules/cm) administered via a fiber diffuser determines the depth of effective NIR-PIT around the diffuser and determines the spacing at which such diffusers should be placed to entirely cover the tumor. Thermal measurements demonstrate that interstitial light for NIR-PIT does not cause damage to the skin overlying the diffuser.

Near Infrared Photoimmunotherapy with Combined Exposure of External and Interstitial Light Sources.

Near infrared photoimmunotherapy (NIR-PIT) is a new target-cell-specific cancer treatment that induces highly selective necrotic/immunogenic cell death after systemic administration of a photoabsorber antibody conjugate and subsequent NIR light exposure. However, the depth of NIR light penetration in tissue (approximately 2 cm) with external light sources limits the therapeutic effects of NIR-PIT. Interstitial light exposure using cylindrical diffusing optical fibers can overcome this limitation. The purpose in this study was to compare three NIR light delivery methods for treating tumors with NIR-PIT using a NIR laser system at an identical light energy; external exposure alone, interstitial exposure alone, and the combination. Panitumumab conjugated with the photoabsorber IRDye-700DX (pan-IR700) was intravenously administered to mice with A431-luc xenografts which are epithelial growth factor receptor (EGFR) positive. One and 2 days later, NIR light was administered to the tumors using one of three methods. Interstitial exposure alone and in combination with external sources showed the greatest decrease in bioluminescence signal intensity. Additionally, the combination of external and interstitial NIR light exposure showed significantly greater tumor size reduction and prolonged survival after NIR-PIT compared to external exposure alone. This result suggested that the combination of external and interstitial NIR light exposure was more effective than externally applied light alone. Although external exposure is the least invasive means of delivering light, the combination of external and interstitial exposures produces superior therapeutic efficacy in tumors greater than 2 cm in depth from the tissue surface.

Activatable fluorescent probes in fluorescence-guided surgery: Practical considerations.

Fluorescence-guided imaging during surgery is a promising technique that is increasingly used to aid surgeons in identifying sites of tumor and surgical margins. Of the two types of fluorescent probes, always-on and activatable, activatable probes are preferred because they produce higher target-to-background ratios, thus improving sensitivity compared with always-on probes that must contend with considerable background signal. There are two types of activatable probes: 1) enzyme-reactive probes that are normally quenched but can be activated after cleavage by cancer-specific enzymes (activity-based probes) and 2) molecular-binding probes which use cancer targeting moieties such as monoclonal antibodies to target receptors found in abundance on cancers and are activated after internalization and lysosomal processing (binding-based probes). For fluorescence-guided intraoperative surgery, enzyme-reactive probes are superior because they can react quickly, require smaller dosages especially for topical applications, have limited side effects, and have favorable pharmacokinetics. Enzyme-reactive probes are easier to use, fit better into existing work flows in the operating room and have minimal toxicity. Although difficult to prove, it is assumed that the guidance provided to surgeons by these probes results in more effective surgeries with better outcomes for patients. In this review, we compare these two types of activatable fluorescent probes for their ease of use and efficacy.

Molecularly Targeted Cancer Combination Therapy with Near-Infrared Photoimmunotherapy and Near-Infrared Photorelease with Duocarmycin-Antibody Conjugate.

Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody-photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab-IR700 (pan-IR700) and the NIR-releasing compound, CyEt-panitumumab-duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line in vitro In in vivo studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor-background ratio. To evaluate the effects of combination therapy in vivo, tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.05), and significantly prolonged survival was achieved (P < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. Mol Cancer Ther; 17(3); 661-70. ©2017 AACR.

Dynamic changes in the cell membrane on three dimensional low coherent quantitative phase microscopy (3D LC-QPM) after treatment with the near infrared photoimmunotherapy.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that relies on the binding of a near-infrared antibody photoabsorber conjugate (APC) to a cancer cell. Subsequent exposure to NIR light selectively induces rapid necrotic cell death on target-expressing cells with minimal off-target effects. When treated with NIR-PIT, targeted cells become swollen, develop blebs and burst within minutes of light exposure. Detailed spatial and temporal morphological changes of the cellular membrane of targeted cells treated with NIR-PIT have not been fully explored with state-of-the-art microscopic methods. In this study, we investigated the morphologic and kinetic effects of PIT on two types of cells, a spindle-shaped 3T3/Her cell and a spheric-shaped MDA-MB468 cell, after NIR-PIT using three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM). Adhesive cells treated with NIR-PIT demonstrated region-specific cell membrane rupture occurring first on the distal free edge of the cell near the site of adhesion, in a process that was independent of cell shape. The results show that the peripheral portions of the cell membrane near the site of adhesion are particularly vulnerable to the effects of NIR-PIT, likely because these sites exhibit higher baseline surface tension.

Evaluation of Early Therapeutic Effects after Near-Infrared Photoimmunotherapy (NIR-PIT) Using Luciferase-Luciferin Photon-Counting and Fluorescence Imaging.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that induces highly selective immunogenic cell death. It is based on an antibody-photoabsorber conjugate (APC) that is activated by NIR light. The purpose of this study was to investigate the effects of NIR-PIT as measured by luciferase-luciferin photon-counting and fluorescence imaging. Six days after subcutaneous injection of A431-luc-GFP cells tumors formed in a xenograft mouse model. The EGFR-targeting antibody, panitumumab, was conjugated to the photoabsorber, IRDye-700DX (pan-IR700), and was intravenously administered to tumor-bearing mice. Serial luciferase-luciferin photon-counting images and both green fluorescent protein (GFP) and IR700 fluorescence images were obtained from the same mice before and after NIR-PIT treatment (0, 10, 20, 30 min (early phase), and 24, 48 h (late phase) after NIR light exposure). Optical signal intensities were compared for each modality. IR700 fluorescence and luciferase-luciferin photon-counting images showed decreased intensities in both the early and late phases after NIR-PIT (p < 0.01). On the other hand, GFP fluorescence images showed decreased intensities only in the late phase (p < 0.01). In the early phase, GFP fluorescence images showed smaller intensity reductions compared to IR700 fluorescence and luciferase-luciferin photon-counting (p < 0.01), while in the late phase, IR700 fluorescence showed smaller intensity reductions than luciferase-luciferin photon-counting and GFP fluorescence (p < 0.05), due to redistribution of pan-IR700 within the tumor bed. In conclusion, luciferase-luciferin photon-counting imaging is suitable to evaluate early phase NIR-PIT effects, while both luciferase-luciferin photon-counting and GFP reflected later phase effects.

Characteristics of ovarian cancer detection by a near-infrared fluorescent probe activated by human NAD(P)H: quinone oxidoreductase isozyme 1 (hNQO1).

Near-infrared (NIR) fluorescent probes are ideal for in vivo imaging, because they offer deeper tissue penetration by the light and lower background autofluorescence than fluorophores that emit in the visible range. Q3STCy is a newly synthesized, NIR light-emitting probe that is activated by an enzyme commonly overexpressed in tumor cells, human nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase isozyme 1, known as hNQO1 or DT-diaphorase. The purpose of this study is to compare the sensitivity of detecting peritoneal ovarian cancer metastasis (POCM) with Q3STCy and gGlu-HMRG, a green fluorescent probe, upon their surface application. In vitro uptake of Q3STCy was significantly higher than that of gGlu-HMRG. Using a red fluorescence protein (RFP)-labeled in vivo tumor model of POCM, the Q3STCy probe provided high sensitivity (96.9%) but modest specificity (61.0%), most likely the result of albumin-probe interactions and non-specific activation in nearby altered but healthy cells. Three types of kinetic maps based on maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC) allowed for differentiation of the activated fluorescence signal associated with POCM from the background signal of the small intestine, thereby significantly improving the specificity of Q3STCy to 80%, 100%, and 100% for MF, WIR, and AUC, as well yielding a moderate improvement in sensitivity (100% for all approaches) that is comparable to that with gGlu-HMRG, but with the added advantages of NIR fluorescence as the transduction modality. Such a new methodology has the potential to afford identification of cancerous lesions deeper within tissue.

Syngeneic Mouse Models of Oral Cancer Are Effectively Targeted by Anti-CD44-Based NIR-PIT.

Oral cavity squamous cell carcinoma (OSCC) is considered one of the most aggressive subtypes of cancer. Anti-CD44 monoclonal antibodies (mAb) are a potential therapy against CD44 expressing OSCC; however, to date the therapeutic effects have been disappointing. Here, a new cancer treatment is described, near-infrared photoimmunotherapy (NIR-PIT), that uses anti-CD44 mAbs conjugated to the photoabsorber IR700DX. This conjugate is injected into mice harboring one of three CD44 expressing syngeneic murine oral cancer cell (MOC) lines, MOC1 (immunogenic), MOC2 mKate2 (moderately immunogenic), and MOC2-luc (poorly immunogenic). Binding of the anti-CD44-IR700 conjugate was shown to be specific and cell-specific cytotoxicity was observed after exposure of the cells to NIR light in vitro The anti-CD44-IR700 conjugate, when assessed in vivo, demonstrated deposition within the tumor with a high tumor-to-background ratio. Tumor-bearing mice were separated into four cohorts: no treatment; 100 µg of anti-CD44-IR700 i.v. only; NIR light exposure only; and 100 µg of anti-CD44-IR700 i.v. with NIR light exposure. NIR-PIT therapy, compared with the other groups, significantly inhibited tumor growth and prolonged survival in all three cell model systems. In conclusion, these data reveal that anti-CD44 antibodies are suitable as mAb-photoabsorber conjugates for NIR-PIT in MOC cells.Implications: This study using syngeneic mouse models, which better model the disease in humans than conventional xenografts, suggests that NIR-PIT with anti-CD44-IR700 is a potential candidate for the treatment of OSCC. Mol Cancer Res; 15(12); 1667-77. ©2017 AACR.

Noninvasive screening test for detecting early stage lymphedema using follow-up computed tomography imaging after cancer treatment and results of treatment with lymphaticovenular anastomosis.

BACKGROUND: Although early detection is valuable for secondary lymphedema treatment, existing screening tests are not popular. This study aimed to propose a novel method of screening lymphedema patients based on the thickness of the subcutaneous fat measured with perioperative computed tomography (CT) and present the results from evaluation of patients who underwent those examinations was performed. METHOD: The medical records of 96 gynecological cancer patients and 189 breast cancer patients, whose lymphatic function was assessed with indocyanine green lymphography, were reviewed. In gynecological cancer patients, the perioperative temporal subcutaneous fat thickness index (T-SFTI) was calculated from presurgical and follow-up CT data, and in breast cancer patients, the postoperative crosswise subcutaneous fat thickness index (C-SFTI) was calculated. In lower extremity lymphedema patients, the effect of lymphaticovenular anastomosis (LVA) was also evaluated using T-SFTI. RESULTS: Perioperative T-SFTI was assessed in 180 lower extremities, and it was significantly higher in 46 lymphatic dysfunction limbs (1.21 ± 0.08) than in 134 normal lymphatic function limbs (1.03 ± 0.08), (P < .01). Postoperative C-SFTI was assessed in 53 upper extremity, and it was significantly higher in 11 lymphatic dysfunction limbs (1.31 ± 0.21) than in 42 normal lymphatic function limbs (1.01 ± 0.06), (P < .01). In lower extremity lymphedema patients, T-SFTI improved significantly after planned conservative treatments and LVA (P = .04). CONCLUSION: Assessment of subcutaneous fat thickness using CT is useful for screening early stage lymphedema. If the efficacy of this method is validated, patients worldwide may be assessed using the same criterion.

Near-Infrared Photoimmunotherapy Targeting Prostate Cancer with Prostate-Specific Membrane Antigen (PSMA) Antibody.

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for molecular therapy. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photoabsorber conjugate (APC). Here, we describe the efficacy of NIR-PIT, using a fully human IgG1 anti-PSMA monoclonal antibody (mAb), conjugated to the photoabsorber, IR700DX, in a PSMA-expressing PC3 prostate cancer cell line. Anti-PSMA-IR700 showed specific binding and cell-specific killing was observed after exposure of the cells to NIR light in vitro In the in vivo study, anti-PSMA-IR700 showed high tumor accumulation and high tumor-background ratio. Tumor-bearing mice were separated into 4 groups: (i) no treatment; (ii) 100 µg of anti-PSMA-IR700 i.v.; (iii) NIR light exposure; (iv) 100 µg of anti-PSMA-IR700 i.v., NIR light exposure was administered. These were performed every week for up to 3 weeks. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other control groups (P < 0.001), and significantly prolonged survival was achieved (P < 0.0001 vs. other control groups). More than two thirds of tumors were cured with NIR-PIT. In conclusion, the anti-PSMA antibody is suitable as an APC for NIR-PIT. Furthermore, NIR-PIT with the anti-PSMA-IR700 antibody is a promising candidate of the treatment of PSMA-expressing tumors and could be readily translated to humans.Implications: NIR-infrared photoimmunotherapy (NIR-PIT) using a fully human anti-PSMA-IR700 conjugate showed potential therapeutic effects against a PSMA-expressing prostate cancer that is readily translated to humans. Mol Cancer Res; 15(9); 1153-62. ©2017 AACR.

Near-infrared photoimmunotherapy: a comparison of light dosing schedules.

Near infrared photoimmunotherapy (NIR-PIT) is a newly-developed cancer therapy in which a monoclonal antibody is conjugated to a near-infrared photoabsorber, IR700 to form an antibody photoabsorber conjugate (APC). After the APC binds to cancer cells expressing the cognate antigen, exposure to NIR light results in rapid, highly selective necrotic cell death of the cancer cells with minimal off-target effects. Several hours after NIR-PIT, the tumor vessels become supraphysiologically permeable and circulating APC can therefore readily leak into the already-treated tumor space where it can bind with viable cancer cells that is called super-enhanced permeability and retention effect. The presence of the SUPR effect after NIR-PIT has prompted regimens in which there is a repeat exposure of NIR light 24 hours after the initial NIR-PIT to take advantage of the leakage of additional APC deeper into the tumor. However, this post-treatment APC penetration was fully induced within 3 hours, therefore, it is possible that repeated exposures of NIR light could be administered much earlier than 24 hours and still produce the same effects. To test this idea, we compared several modes of delivering additional doses of light after initial NIR-PIT. We found that repeated exposures of NIR light starting 3 hours after initial NIR-PIT produced equal or superior results to more delayed exposures of NIR light. This finding has practical implications of an easy-to-perform regimen as repeated light exposures could be performed during a single day rather than extending the procedure over two days which is the current recommendation.

A topically-sprayable, activatable fluorescent and retaining probe, SPiDER-ßGal for detecting cancer: Advantages of anchoring to cellular proteins after activation.

SPiDER-ßGal is a newly-developed probe that is activated by ß-galactosidase and is then retained within cells by anchoring to intracellular proteins. Previous work has focused on gGlu-HMRG, a probe activated by γ-glutamyltranspeptidase, which demonstrated high sensitivity for the detection of peritoneal ovarian cancer metastases in an animal model. However, its fluorescence, after activation by γ-glutamyltranspeptidase, rapidly declines over time, limiting the actual imaging window and the ability to define the border of lesions. The purpose of this study is to compare the fluorescence signal kinetics of SPiDER-ßGal with that of gGlu-HMRG using ovarian cancer cell lines in vitro and ex vivo tissue imaging. In vitro removal of gGlu-HMRG resulted in a rapid decrease of fluorescence intensity followed by a more gradual decrease up to 60 min while there was a gradual increase in fluorescence up to 60 min after removal of SPiDER-ßGal. This is most likely due to internalization and retention of the dye within cells. This was also confirmed ex vivo tissue imaging using a red fluorescence protein (RFP)-labeled tumor model in which the intensity of fluorescence increased gradually after activation of SPiDER-ßGal. Additionally, SPiDER-ßGal resulted in intense enhancement within the tumor due to the high target-to-background ratio, which extended up to 60 min after activation. In contrast, gGlu-HMRG fluorescence resulted in decreasing fluorescence over time in extracted tumors. Thus, SPiDER-ßGal has the advantages of higher signal with more signal retention, resulting in improved contrast of the tumor margin and suggesting it may be an alternative to existing activatable probes.

Cerenkov Radiation-Induced Photoimmunotherapy with 18F-FDG.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with toxicity induced by photoabsorbers after irradiation with NIR light. A limitation of NIR-PIT is the inability to deliver NIR light to a tumor located deep inside the body. Cerenkov radiation (CR) is the ultraviolet and blue light that is produced by a charged particle traveling through a dielectric medium faster than the speed of light in that medium and is commonly produced during radioactive decay. Here, we demonstrate the feasibility of using CR generated by 18F-FDG accumulated in tumors to induce photoimmunotherapy. Methods: Using A431-luc cells, we evaluated the therapeutic effects of CR-PIT in vitro and in vivo using bioluminescence imaging. Results: CR-PIT showed significant suppression of tumor size, but the decrease of bioluminescence after CR-PIT was not observed consistently over the entire time course. Conclusion: Although CR-PIT can induce tumor killing deep within body, it is less effective than NIR-PIT, possibly related to the relatively lower efficiency of short wavelength light than NIR.

A heart-brain-kidney network controls adaptation to cardiac stress through tissue macrophage activation.

Heart failure is a complex clinical syndrome characterized by insufficient cardiac function. In addition to abnormalities intrinsic to the heart, dysfunction of other organs and dysregulation of systemic factors greatly affect the development and consequences of heart failure. Here we show that the heart and kidneys function cooperatively in generating an adaptive response to cardiac pressure overload. In mice subjected to pressure overload in the heart, sympathetic nerve activation led to activation of renal collecting-duct (CD) epithelial cells. Cell-cell interactions among activated CD cells, tissue macrophages and endothelial cells within the kidney led to secretion of the cytokine CSF2, which in turn stimulated cardiac-resident Ly6Clo macrophages, which are essential for the myocardial adaptive response to pressure overload. The renal response to cardiac pressure overload was disrupted by renal sympathetic denervation, adrenergic ß2-receptor blockade or CD-cell-specific deficiency of the transcription factor KLF5. Moreover, we identified amphiregulin as an essential cardioprotective mediator produced by cardiac Ly6Clo macrophages. Our results demonstrate a dynamic interplay between the heart, brain and kidneys that is necessary for adaptation to cardiac stress, and they highlight the homeostatic functions of tissue macrophages and the sympathetic nervous system.