Browsing by Author "Palmer, Gregory M"

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Approximately half of bladder cancer diagnoses are late stage/show metastatic spread. The current standard treatment option for these diagnoses is cisplatin chemotherapy, however 40% of patients are ineligible to receive this treatment, leaving few alternatives. The most common alternative to chemotherapy that is currently used is immune checkpoint blockade or immunotherapy. However, immunotherapy alone has proven largely ineffective as a curative treatment option for the metastatic disease, indicating a critical need for new systemic treatment options. Our group has previously demonstrated in a pilot study the efficacy of a novel combination therapy incorporating two treatment arms: clinically available immunotherapy and gold nanostar mediated photothermal therapy (abbreviated as PTT). This treatment is called Synergistic Immuno Photothermal Nanotherapy (abbreviated as SYMPHONY). In this study, C57BL/6 mice were implanted with MB49 bladder cancer cells at two locations. One of the two subsequent tumors was treated with one of five total treatment arms. After treatment, one of the mice treated with SYMPHONY showed total tumor local control and no tumor regrowth upon MB49 cell rechallenge, suggesting long term cancer immunity induced by SYMPHONY treatment. Currently, the treatment paradigm for SYMPHONY follows as: 1) Gold nanostar (abbreviated as GNS) injection followed by a 24-hour waiting period 2) Mice are put under anesthesia and laser irradiation of the tumor is performed at a constant power density for 10 minutes 3) Anti-PD-L1 immunotherapy injections are given following irradiation. For SYMPHONY’s photothermal therapy treatment arm, quantitative characterization of the GNS photothermal effect and a photothermal treatment planning process are crucial aspects of SYMPHONY that remain undeveloped. The overall goal of SYMPHONY is to provide a systemic, curative treatment option for patients diagnosed with late-stage bladder cancer. The objective of this work is to characterize the photothermal effect as well as develop and validate a photothermal treatment planning workflow to be used in SYMPHONY treatment that accurately predicts thermal dose delivery. The goals of this work were completed using a variety of materials and methods, including: In vitro agarose-GNS gel phantoms along with thermocouples for thermal dosimetry, radiative transport and heat transfer software used to create photothermal simulations, a multitude of characterization methods to extract key GNS physical properties, as well as image segmentation and model generation techniques to analyze animal images and create simulation models. Results from in vitro phantom heating studies and their accompanying photothermal simulations are presented. Additionally, optical absorption characterization data is presented to serve as the basis for selected optical modeling parameters. Trends in GNS-mediated photothermal effect and the dependence on concentration are discussed, as well as their implications for future SYMPHONY studies.

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Background: PD-L1 (Programmed Death Ligand 1) is an immune checkpoint molecule that is commonly expressed on the surface of cancer cells. When it interacts with its receptor – the PD-1 molecule, which is commonly expressed on the surface of immune cells, such as T-cells, it will then deliver a negative signal which in turn inactivates the T-cell function, depresses the T-cell expansion, and dampens the overall tumor response. Anti-PD-L1 antibody blocks the direct interaction between the PD-L1 and PD-1 molecules, thus inhibits the PD-L1 signaling pathways, enabling the immune system, and hence the anti-tumor immunity, to eliminate the cancer cells. We refer to these mechanisms as the Immune Checkpoint Blockade (ICB).

Methods and Materials: In the current study, we report the effects of combination treatment of radiation and ICB on tumor metastasis. A single radiation dose of 10 Gy was used to irradiate the dorsal fat pad region of mice, into which the E0771-luc breast cancer cells were injected. For the ICB treatment, anti-PD-L1 antibody was used. We established a spontaneous metastatic model using E0771-luc breast cancer cells. As the tumor grew, primary tumor growth was monitored using calipers. We then examined the metastatic lung lesions by using In-Vivo Imaging System (IVIS), the signals from IVIS were then quantified in terms of total photon flux (photons/second). An India-Ink Assay was also employed to further verify the lung mass formations. In addition, variation of primary tumors’ hemoglobin saturation levels and total hemoglobin levels were recorded before and after treatments using zenascope, for the sake of assessing the vascular oxygenation and vascularity information, respectively.

Principal Findings: Data from calipers’ measurement showed that the RT-alone group did not show any effects on tumor growth. Tha anti-PD-L1-alone group showed a slightly delayed tumor growth. The combination treatment showed that 3 out of 5 mice showed tumor growth delay, while the tumor regrew after 20 days. Furthermore, survival curves indicated that there is no significant difference among all groups, indicating that radiation treatment or anti-PD-L1 antibody or the combination of both treatments did not affect the time at which the mice reach their endpoints (tumor volume ≥ 1500mm3). In addition, data from the IVIS indicated that the total photon flux emitted from primary tumors varied dramatically among experimental subjects within the same group. There were also extremely low or no luciferase signals from the lung. Statistically, two-way ANOVA for IVIS data showed that there are no significant differences between the RT-alone or anti-PD-L1-alone or RT + anti-PD-L1 group and the control group, for both primary tumors and lung tissue, suggesting that all kinds of treatments used in the current study neither helped eliminate the primary tumor cells nor reduced the burden of metastatic cancer cells in lungs, compared to the control group. Interestingly, results from the India-ink Assay showed that grossly visible lung nodules were not observed in all lungs of the mice, suggesting that the primary tumors in the dorsal fat pad region did not result in grossly visible lung metastases in any groups. Furthermore, analysis for the Zenascope data showed that there was a gradual increase in Hb-Sat(%) in mice for the control group, while a gradual decrease in Hb-Sat(%) for the anti-PD-L1-alone group. The RT-alone group did not show a clear response of change in Hb-Sat. For the combination treatment group, 3 out of 4 mice demonstrated a relatively flat response of change in Hb-Sat(%). Lastly, the total Hb levels in the control group, the anti-PD-L1-alone group and the combination treatment group remained relatively stable over the treatment time. For the RT-alone group, 3 out of 5 mice showed almost no changes, while the other two demonstrated a huge increase in total Hb levels on day 0, and day 2, respectively, but the levels went back to almost the pre-treatment values after day 3.

Conclusions: The combination treatment of 10 Gy of radiation and anti-PD-L1 antibodyimmunotherapy did not show significant effects on E0771 primary tumor growth when using an orthotopic tumor model. The time required for the tumor volume to exceed an endpoint of 1500mm3 was not significantly affected by all of the treatment methods used in the current study. The results from the IVIS and the India-ink Assay suggest that E0771 might not a good model for lung metastasis. However, the treatment response and the E0771 model were affected by a number of technical problems that render the evaluation inconclusive. Solutions to some of these technical problems have been provided, enabling future reseachers to replicate and improve on this study and futher determine the treatment response and the usefulness of the E0771 model. Regarding the zenascope measurement, the changes in Hb-Sat(%) may be correlated with the blood vessel growth within the primary tumors, while the changes in total Hb were almost negligible. Nevertheless, several limitions when performing the zenascope measurement have been listed, including the pigmentation and fur of the skin of mice, the motion of the mice and/or the operator’s hands, as well as the uncertainties in placing the optical probe onto the tumor. Further research is needed to uncover the promise of this combined therapy and to further verify the correlation among the changes in Hb-Sat(%), the changes in total Hb levels, and the tumor physiological characteristics.

Hypoxia is one of the primary causes of radioresistance in human solid tumors. There are a series of contributing factors such as blood-flow rate, oxygen consumption rate, arterial pO2, and microvascular arrangement. In this study, blood-flow rate, oxygen consumption rate (OCR), and arterial pO2 are simulated to analyze their effects on hypoxia. A Green’s function method is applied in a densely vascularized tumor region to predict oxygen delivery. The results indicate that changes in OCR lead to greater changes in hypoxia. A 30% reduction in oxygen consumption rate leads to a 21% reduction in the hypoxic fraction. However, a 30% increase in blood-flow rate and arterial pO2 results in a 6% and 13% reduction in hypoxic fraction respectively. A 20% reduction in oxygen consumption rate plus a 20% increase in arterial pO2 causes a 40% decrease in hypoxic fraction. With increasing blood-flow rate and arterial pO2, hypoxic fraction reaches a plateau. As a result, hypoxia is more sensitive to OCR. In the second phase of this work, modeling results were compared to experimental data indicating the effect of papaverine in modulating tumor hypoxia. Papaverine is an FDA (Food and Drug Administration)-approved drug that can effectively decrease oxygen consumption rate and thus potentially decrease hypoxic fraction (~14%) at 2 mg/kg. At 4mg/kg, the addition of papaverine did not lead to a decrease in hypoxic fraction. This result fits the hypothesis that the oxygen consumption rate may be balanced by the effects of vasodilation which may induce more tumor shunting and poor perfusion. However, the results shown here are limited to few animals. A larger number of animals in each group, combined with other types of evidence such as perfusion staining is conducive to get a more accurate result and a better understanding of the underlying mechanism. Therefore, these experiments can be regarded as preliminary results and suggest opportunities for future experimental work.

Purpose

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Results

Conclusions

Cancer is a significant threat to human health with more than eight million deaths each year in the world. Therefore, numerous technologies have been implemented or under development to effectively treat cancer.

One novel therapeutic platform is implemented using nanoparticle-mediated photothermal therapy. Gold NanoStars (GNS), are a unique form of gold nanoparticles (GNPs) that have unique therapeutic potential because of their star-shaped geometry. Enhanced light absorption and higher photon-to-heat conversion efficiency are introduced by GNS’s plasmonic properties. In the application of hyperthermia, this photothermal process can be exploited to specifically heat tumors and, more importantly, to amplify the antitumor immune response following the highly immunogenic thermal death of cancer cells. Meanwhile, when combined with immune checkpoint inhibition immunotherapy (IT), this SYnergistic iMmuno PHOtothermal NanotherapY (SYMPHONY) has been shown to reverse tumor-mediated immunosuppression, thereby leading to the treatment of not only primary tumors but also cancer metastasis. This phenomenon is called the “abscopal effect”. However, the immune response has not been clearly quantified yet. Our hypothesis was that different treatment modalities (PTT only, GNS-PTT, IT, SYMPHONY) will trigger different levels of immune response including the decrease of immunosuppressive cells and the influx of cytotoxic cells, which could be observed by imaging immune cell reporters.

Accordingly, two specific aims were set for this study: 1. to develop a pre-clinical murine model to quantify different levels of immune response mimicking the tumor metastatic environment; 2. to quantify immune response following SYMPHONY using imaging analysis techniques.

To achieve the specific aims, window chamber models combined with in vivo fluorescence imaging techniques provide an ideal platform to mimic cancer metastasis in the chamber and longitudinally monitor immune response through the prevalence of fluorescent reporters specifically localized to immune cells of interest. We utilized a dual tumor mouse model, consisting of a primary tumor grown in the flank of the mouse which received the SYMPHONY therapy, as well as a secondary tumor located in the window chamber through which we could image and observe the abscopal response to therapy. In this study, we demonstrate the optical imaging procedure following synergistic immune photothermal therapy (SYMPHONY) of bladder cancer using the immune-GFP-labeled murine window chamber model, for the purpose of quantifying the immune response at this mimicked-tumor metastasis site. Four groups were established: the SYMPHONY group, the photothermal therapy group, the immune therapy group, and the Gold NanoStars (GNS) control group.

Higher immune responses were observed in the tumor regions compared to the non-tumor regions. The in vivo fluorescence imaging along with the window chamber technique demonstrates the feasibility and convenience of following such a longitudinal study like SYMPHONY. However, although temporal changes in reporter intensity were observed, with a limited number of samples, we cannot thus far identify significant differences among the treatment groups. Approaches for further characterizing this model are discussed.

In vivo imaging is an important scientific tool for studying bio-molecular interactions, but lack of preserved functionality during imaging restricts scientists’ abilities to gain critical knowledge. Structure can be preserved while using high-resolution optical imaging by utilizing window chambers in murine models1, yet the use of anesthesia for immobilization is problematic. Anesthesia affects tissue oxygenation2, blood cell velocities3, immunosuppression4, and allowable duration of imaging5–thus its usage restricts in vivo bio-molecular imaging accuracy and duration.

Developing a portable imaging system that attaches to murine dorsal window chambers enables imaging without anesthesia, avoiding previous drawbacks of window chamber models. A raspberry pi camera (RPI-CAM-V2, Raspberry Pi) was modified for microscopy and used alongside 3D printed panels for attaching the camera, optical filters, and LED light source to murine window chambers. Multiple applications for the portable system were developed, each requiring their own setup of filters and stimulating LEDs. The system is powered by a Raspberry Pi 3 Model B single-board computer (RASPBERRYPI3-MODB-1GB, Raspberry Pi), allowing for stream-lined data acquisition.

Imaging tissue oxygenation was the first application developed for the portable system. Oxygen sensing boron nanoparticles were injected into window chambers, while a UV LED was used to stimulate fluorescent and phosphorescent signals. When stimulated by UV light, the boron nanoparticles emit fluorescence and phosphorescence. Fluorescence is stable regardless of oxygenation, while phosphorescence signal from the nanoparticles is quenched in the presence of higher oxygenations. The ratio of fluorescence to phosphorescence was used to calculate oxygen concentration maps of window chamber tissue. Tissue oxygenations in awake and anesthetized mice inhaling varied oxygen concentrations were analyzed. In 5 awake nude mice inhaling 20% O2, the median partial pressure of oxygen was measured as 49 mmHg within their window chambers. From a one-tailed t-test with a false positive correction, 3 of the mice had significantly higher (p ≤ 0.05) tissue oxygenation while anesthetized compared to the awake measurements.

Developing the portable systems ability to image blood cells was another focus of this project. Blood cells were visible with white LED exposure. A frame rate of 30 frames/second was adequate for tracking cell motion while allowing for the highest resolution possible with the system. Blood cell velocities in a mouse awake and anesthetized were analyzed, while also observing change in blood cell velocities during sepsis that was induced by cecal ligation puncture (CLP). Three days after CLP, the mean awake blood cell velocity was measured as 0.21 ± 0.03 mm/s, while the mean anesthetized blood cell velocity was measured as 0.080 ± 0.002 mm/s. Six days after CLP, the awake measurement had reduced to 0.019 ± 0.005 mm/s, while the anesthetized measurement was reduced to 0.031 ± 0.002 mm/s (91% decrease in awake measurement, 61% decrease in anesthetized measurement). A two-way ANOVA on the factors of anesthesia and time post-CLP performed on multiple vessel regions calculated significance (p ≤ 0.05) for both of these factors on blood cell velocities within the pilot mouse’s window chamber.

Noting the differences between data collected on awake and anesthetized mice, our system has been validated as a tool for real-time imaging of tissue without the observed effects of anesthesia. By avoiding anesthesia, the developed device allows for continual data acquisition to increase from hours to days. The system is generalizable, and while only two applications are presented in this study, the system could be modified for imaging fluorescently labeled cells/proteins for other bio-molecular interactions.

Bladder cancer is a malignant tumor that develops on the mucosa of the bladder. It is one of the top ten occurring cancers in the body and one of the top ten malignant tumors in the urinary system. Non-muscle invasive bladder cancer is the most common type of bladder cancer, accounting for approximately 70% of all patients with bladder cancer. Typically, for patients who are detected to have muscle-invasive bladder cancer, the hope of cure is less than for patients who have non-muscle-invasive bladder cancer. Nowadays, chemotherapy based on cisplatin is the most common treatment for metastatic bladder cancer. However, only about half of bladder cancer patients are suitable for chemotherapy, and its efficacy ranges from 40% to 65%. Immunotherapy, which reactivates the inhibitory function of immune cells by blocking immunological checkpoints for the aim of treating cancer, is the most generally considered alternative to chemotherapy at present. Immune activation using BCG has also been proposed as a viable treatment for bladder cancer. Yet, these are insufficient to prove effective against metastatic cancers. SYnergistic iMmuno PHOtothermal NanotherapY, called SYMPHONY, refers to the combination of immunotherapy and gold nanostar-mediated photothermal therapy for primary tumor. Gold nanostar-mediated photothermal therapy is the use of a wavelength of 1064 nm laser irradiation for about 10 minutes in the mouse flank tumor location, and its treatment is local. Unlike photothermal treatment, the immune response induced by anti-PD-L1 injection is systemic. We believe that this therapy can enhance the clinical treatment of bladder cancer with metastasis. In previous investigations, C57BL/6 mice were injected at two locations with MB49 bladder cancer cells, but only one location received local laser treatment with 1064 nm wavelength. Tumor volume over time was recorded. One of the five SYMPHONY mice was able to suppress the tumor following treatment, however, the other groups were unable to reach the same outcome. When the same tumor cells were utilized to challenge the mice a second time, the tumor exhibited no symptoms of development, demonstrating a long-term immunity has occurred. The cure for metastatic bladder cancer is related to the development of an immune memory response, as this concept has been prooven. Yet, there is still little known about quantification of the immune response at the distant tumor location after treatment. Our long-term goal is to develop an effective alternative treatment for patients with metastatic bladder cancer. The overall objective of this application is to quantify the immune responses of transgenic mice with fluorescent report genes on myeloid cells (monocytes, macrophages, natural killer cells, dendritic cells), and T cells undergoing one of four treatments (no treatment (control), nano-photothermal therapy (GNS), immunotherapy (anti-PD-L1), SYMPHONY)), and the time point for additional research within the week after treatment was determined. Our central hypothesis is that transgenic mice treated with SYMPHONY would show higher immune cell infiltration in distant tumors within 48 hours after treatment, and symphony-induced immune response in distant tumors would be stronger than antipdl1 alone. This hypothesis was tested by implanting a primary flank tumor and an untreated small distant tumor in 20 mice. The distant tumor cells were stained with DiD red fluorescent dye and then injected into the center of a dorsal skinfold window chamber. The dorsal skinfold window chamber is on the skin of the back of the mouse sutured by surgery for imaging. The primary tumors received one of the four treatments and were imaged using intravital microscope over the 7 days after treatment. From this study, we found that the immune signal of macrophages in the SYMPHONY treatment group peaked on the third day of treatment and then decreased continuously, which was consistent with the change trend of the window chamber tumor area. Secondly, and the primary tumor volume and distant window chamber tumor area decreased within seven days of SYMPHONY treatment group. This is supportive of our hypothesis and previously published work. Future research should concentrate on the immunological response of mice on the day of treatment, monitor the changes in multiple immune cells, and quantify the effects of laser treatment on this day. Notably, the use of immune signals around distant tumor to predict immune response within tumor requires imaging at intervals more frequently than every 24 hours, because estimating the rate of macrophage movement in tissues, where the more appropriate imaging frequency is every 2 hours.

Radiation therapy is a frequently used treatment method for malignant tumors despite the heterogeneous response in tumors with a hypoxic microenvironment. Specific features in this microenvironment like poorly formed and inefficient vasculature contribute to chronic and cycling hypoxia. Notably, hypoxic tumor cells are three times more radioresistant than normoxic cells, which make hypoxia a key contributor to poor treatment outcome. There have been many previous attempts to re-oxygenate tumors either through increasing the supply or decreasing the demand of oxygen. However, no study has yet been performed to investigate the combined effect of increasing the oxygen supply and decrease the oxygen demand in vivo.

There are two main purposes of this study, which are tested in two individual rounds: 1) assessing the combined effect of oxygen micro-bubbles and papaverine in alleviating tumor hypoxia in murine sarcoma model and 2) assessing the combined effect of oxygen micro bubble and papaverine as radiosensitizers. Using nu-nu mice with subcutaneous sarcoma tumors, the change resulting from oxygen micro-bubbles and/or papaverine was evaluated by changes in hemoglobin saturation from baseline and control groups. By further monitoring tumor growth and percent hemoglobin saturation after administration of papaverine and oxygen micro-bubbles followed by a single fraction of 15 Gy of radiation, we also tested the effects of the combination of papaverine and oxygen micro-bubbles in tumor control and oxygenation.

The result of the non-irradiated study showed no significant improvement of the combination of oxygen micro-bubbles and papaverine group in the percent hemoglobin saturation level compared with other groups. Notably, percent hemoglobin saturation changes are rather heterogenous within each group. However, this unexpected result may be due to certain practical and theoretic limitations. The follow-up immunohistochemistry study may provide more information of the overall oxygenation of the tumors. For the irradiated study, the percent hemoglobin saturation measurement of the oxygen micro-bubbles and papaverine group is the only one showed improved level the day after the treatment compared with the day before treatment. The combination of oxygen micro-bubbles and papaverine did not show increased tumor control after radiotherapy compared with other groups. However, it should be noted that there are some practical and theoretical limitations in the study that may have contributed in this which is discussed in detail in the discussion chapter. Further studies might be needed to investigate the reasons for this unexpected result.

Hypoxic tumor microenvironments have a clear correlation with a lack of radiosensitivity and diminished therapy response. This relationship can be described through the use of fluorescent and phosphorescent nanoparticles optically imaged in a mouse model. Through the use of this ratiometric oxygen sensing, the hypoxic state of the cancerous tumor can be compared. Normally, the microscope imaging system requires the mouse to be imaged under anesthesia and data recorded for a short amount of time. This has led to challenges in clearly defining the oxygen saturation levels in hemoglobin because the anesthesia can affect the tumor vascular dynamics. Moreover, it is desirable to track blood flow and oxygenation changes over a longer period of time in order to characterize the dynamics of cycling hypoxia and therapeutic response. Therefore, a mobile imaging apparatus has been designed and built to directly attach to the dorsal skinfold window chamber installed on nude murine models. Current progress includes quantifiable ratiometric oxygenation in boron nanoparticle solutions imaged under UV light with the mobile unit. The concept has also been successful in in vivo studies for anesthetized mice. The mobile unit is capable of resolving vasculature and is sensitive enough to record nanoparticle emissions originating from tissue in a mouse window chamber model. This system will use dynamic microscopy to image the tumor’s hypoxic environment on un-anesthetized mice and yield insight into tumor biology and therapeutic response.

Bladder cancer has been ranked as one of the top ten and top twenty most commonly occurring cancers in men and women, respectively, with approximately half of the diagnoses being late stage and/or metastatic disease. The current standard-of-care treatment for metastatic bladder cancer is cisplatin-based chemotherapy, but only about 60% of patients qualify for this treatment option and the remaining cohort have few alternatives available. In fact, the only widely considered alternative is immune checkpoint blockades, or immunotherapies, which work to reactivate inhibited functions of immune cells. Unfortunately, these alone have not proven effective against metastatic malignancies. We believe that we can enhance the effects of clinically available immunotherapies with the addition of nanostar mediated photothermal therapy to the primary tumor. In fact, this combination of anti-PD-L1 immune checkpoint blockade and gold nanostar mediated photothermal therapy, henceforth called SYnergistic iMmuno PHOtothermal NanotherapY (SYMPHONY), was previously tested in a pilot study where C57BL/6 mice were injected with MB49 bladder cancer cells at two locations. One of the sites was treated with one of five treatments and the second remained untreated. Both tumor volumes were measured over time and the survival of the mice was also documented. This study resulted in one of the five SYMPHONY mice having complete tumor control after treatment and no other treatment group had this outcome. Upon rechallenge of the same tumor cell line, a tumor did not grow suggesting long-term immunity to this cancer. Along with a proof of concept, these studies were successful in identifying that macrophages and T-cells are associated with the tumor eradication. However, there is still little known about the quantification of the immune response at the distant tumor site after treatment. Our long-term goal is to develop an effective alternative treatment option for patients with metastatic bladder cancer. The overall objective of this application was to quantify the immune response of transgenic mice with fluorescent reporter genes on monocytes, natural killer cells, and dendritic cells undergoing one of four treatments (SYMPHONY, photothermal therapy alone (GNS), immunotherapy alone (anti-PD-L1) or no treatment (control)) as well as identify time points within the week following therapy in which additional studies could be conducted. Our central hypothesis was that mice treated with SYMPHONY would exhibit an elevated immune cell infiltration at the site of the distant tumor within ~48 hours post treatment, and that SYMPHONY will induce a greater immune response at the distant tumor site compared to anti-PD-L1 alone. This hypothesis was tested by implanting a primary flank tumor and a smaller, untreated distant tumor in 14 mice. The distant tumor cells were first stained with a far-red DiD fluorescent dye before injection into the center of a dorsal skinfold window chamber. The primary tumors were treated with one of the four treatment modalities and the window chamber was imaged using intravital microscopy for 7 days after the treatment. From this study, we found that the greatest change in the immune signal for any group occurred in the SYMPHONY group on the day of treatment and this immune signal remained elevated throughout the 7-day imaging period. This change was greater than all other treatment groups, including the anti-PD-L1 group, therefore, we accept our hypothesis. Further work should focus on assessing the immune response in mice on the day of treatment, looking at other immune cell types and by quantifying the effects of laser treatment on this day. It is noted that using the immune signal surrounding the distant tumor to predict an immune response within the tumor requires imaging at intervals more frequent than every 24 hours because of estimated macrophage travel velocity in tissue, where a more suitable frequency would be every 1.5-2 hours.