NIR-guided dendritic nanoplatform for improving antitumor efficacy by combining chemo-phototherapy.

BACKGROUND: Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), is a promising noninvasive strategy in the treatment of cancers due to its highly localized specificity to tumors and minimal side effects to normal tissues. However, single phototherapy often causes tumor recurrence which hinders its clinical applications. Therefore, developing a NIR-guided dendritic nanoplatform for improving the phototherapy effect and reducing the recurrence of tumors by synergistic chemotherapy and phototherapy is essential. METHODS: A fluorescent targeting ligand, insisting of ICG derivative cypate and a tumor penetration peptide iRGD (CRGDKGPDC), was covalently combined with PAMAM dendrimer to prepare a single agent-based dendritic theranostic nanoplatform iRGD-cypate-PAMAM-DTX (RCPD). RESULTS: Compared with free cypate, the resulted RCPD could generate enhanced singlet oxygen species while maintaining its fluorescence intensity and heat generation ability when subjected to NIR irradiation. Furthermore, our in vitro and in vivo therapeutic studies demonstrated that compared with phototherapy or chemotherapy alone, the combinatorial chemo-photo treatment of RCPD with the local exposure of NIR light can significantly improve anti-tumor efficiency and reduce the risk of recurrence of tumors. CONCLUSION: The multifunctional theranostic platform (RCPD) could be used as a promising method for NIR fluorescence image-guided combinatorial treatment of tumor cancers.

Cryo-thermal therapy elicits potent anti-tumor immunity by inducing extracellular Hsp70-dependent MDSC differentiation.

Achieving control of metastatic disease is a long-sought goal in cancer therapy. Treatments that encourage a patient's own immune system are bringing new hopes in reaching such a goal. In clinic, local hyperthermia and cryoablation have been explored to induce anti-tumor immune responses against tumors. We have also developed a novel therapeutic modality of cryo-thermal treatment by alternating liquid nitrogen (LN2) cooling and radio frequency (RF) heating, and better therapeutic effect was achieved in treating metastatic cancer in animal model. In this study, we investigated the mechanism of systemic immune response elicited by cryo-thermal therapy. In the 4T1 murine mammary carcinoma model, we found that local cryo-thermal therapy resulted in a considerable reduction of distant lung metastases, and improved long-term survival. Moreover, results of tumor re-challenge experiments indicated generation of a strong tumor-specific immune memory after the local treatment of primary tumors. Our further study indicated that cryo-thermal therapy caused an elevated extracellular release of Hsp70. Subsequently, Hsp70 induced differentiation of MDSCs into mature DCs, contributing to the relief of MDSCs-mediated immunosuppression and ultimately the activation of strong anti-tumor immune response. Our findings reveal new insight into the mechanism of robust therapeutic effects of cryo-thermal therapy against metastatic cancers.

Interleukin-6 Induced "Acute" Phenotypic Microenvironment Promotes Th1 Anti-Tumor Immunity in Cryo-Thermal Therapy Revealed By Shotgun and Parallel Reaction Monitoring Proteomics.

Cryo-thermal therapy has been emerged as a promising novel therapeutic strategy for advanced breast cancer, triggering higher incidence of tumor regression and enhanced remission of metastasis than routine treatments. To better understand its anti-tumor mechanism, we utilized a spontaneous metastatic mouse model and quantitative proteomics to compare N-glycoproteome changes in 94 serum samples with and without treatment. We quantified 231 highly confident N-glycosylated proteins using iTRAQ shotgun proteomics. Among them, 53 showed significantly discriminated regulatory patterns over the time course, in which the acute phase response emerged as the most enhanced pathway. The anti-tumor feature of the acute response was further investigated using parallel reaction monitoring target proteomics and flow cytometry on 23 of the 53 significant proteins. We found that cryo-thermal therapy reset the tumor chronic inflammation to an "acute" phenotype, with up-regulation of acute phase proteins including IL-6 as a key regulator. The IL-6 mediated "acute" phenotype transformed IL-4 and Treg-promoting ICOSL expression to Th1-promoting IFN-γ and IL-12 production, augmented complement system activation and CD86(+)MHCII(+) dendritic cells maturation and enhanced the proliferation of Th1 memory cells. In addition, we found an increased production of tumor progression and metastatic inhibitory proteins under such "acute" environment, favoring the anti-metastatic effect. Moreover, cryo-thermal on tumors induced the strongest "acute" response compared to cryo/hyperthermia alone or cryo-thermal on healthy tissues, accompanying by the most pronounced anti-tumor immunological effect. In summary, we demonstrated that cryo-thermal therapy induced, IL-6 mediated "acute" microenvironment shifted the tumor chronic microenvironment from Th2 immunosuppressive and pro-tumorigenic to Th1 immunostimulatory and tumoricidal state. Moreover, the magnitude of "acute" and "danger" signals play a key role in determining the efficacy of anti-tumor activity.

A distinct metabolic signature of human colorectal cancer with prognostic potential.

PURPOSE: Metabolic phenotyping has provided important biomarker findings, which, unfortunately, are rarely replicated across different sample sets due to the variations from different analytical and clinical protocols used in the studies. To date, very few metabolic hallmarks in a given cancer type have been confirmed and validated by use of a metabolomic approach and other clinical modalities. Here, we report a metabolomics study to identify potential metabolite biomarkers of colorectal cancer with potential theranostic value. EXPERIMENTAL DESIGN: Gas chromatography-time-of-flight mass spectrometry (GC-TOFMS)-based metabolomics was used to analyze 376 surgical specimens, which were collected from four independent cohorts of patients with colorectal cancer at three hospitals located in China and City of Hope Comprehensive Cancer Center in the United States. Differential metabolites were identified and evaluated as potential prognostic markers. A targeted transcriptomic analysis of 29 colorectal cancer and 27 adjacent nontumor tissues was applied to analyze the gene expression levels for key enzymes associated with these shared metabolites. RESULTS: A panel of 15 significantly altered metabolites was identified, which demonstrates the ability to predict the rate of recurrence and survival for patients after surgery and chemotherapy. The targeted transcriptomic analysis suggests that the differential expression of these metabolites is due to robust metabolic adaptations in cancer cells to increased oxidative stress as well as demand for energy, and macromolecular substrates for cell growth and proliferation. CONCLUSIONS: These patients with colorectal cancer, despite their varied genetic background, mutations, pathologic stages, and geographic locations, shared a metabolic signature that is of great prognostic and therapeutic potential.

Study of thermal effect on breast tumor metabolism and growth using metabonomics.

In this study, the biological effects of long-term mild hyperthermia treatment on tumor metabolism and growth were investigated using 4T1 murine mammary carcinoma, a common animal model of metastatic breast cancer. Periodic thermal treatment (12 hours per day) was applied to tumors and carried out for 3 days, 7 days, 14 days, and 21 days, respectively. The metabolites of tumor tissues were analyzed by gas chromatography-mass spectrometry. The results showed that the growth rate of thermally treated tumors was inversely related to the abundance of long chain fatty acids and acyl glycerols identified in tumor tissues. In the first two weeks, the growth of thermally treated tumors was significantly inhibited, while there was an obvious accumulation of long chain fatty acids and acyl glycerols in tumor tissues. In the third week, the thermally treated tumors adapted to the thermal environment and started to regrow, while the abundance of long chain fatty acids and acyl glycerols decreased in the tumor tissues. These observations suggested that the blockade of long chain fatty acid synthesis during mild hyperthermia treatment of tumors could improve the long-term treatment effect by limiting the supply of substance and energy for tumor re-growth.

Study of tumor growth under hyperthermia condition.

The new concept of keeping primary tumor under control in situ to suppress distant foci sheds light on the treatment of metastatic tumor. Hyperthermia is considered as one of the means for controlling tumor growth. To simulate the tumor growth, a continuum mathematical model has been introduced. The newest understanding of the Warburg effect on the cellular metabolism and diffusion of the nutrients in the tissue has been taken into consideration. The numerical results are compared with the in vivo experimental data by fitting the tumor cell doubling time/tumor cell growth rate under different thermal conditions. Both the tumor growth curve and corresponding average glucose concentration have been predicted. The numerical results have quantitatively illustrated the controlling effect on tumor growth under hyperthermia condition in the initial stage.

A novel thermal treatment modality for controlling breast tumor growth and progression.

The new concept of keeping primary tumor under control in situ to suppress distant foci sheds light on the novel treatment of metastatic tumor. Hyperthermia is considered as one of the means for controlling tumor growth. In this study, a novel thermal modality was built to introduce hyperthermia effect on tumor to suppress its growth and progression using 4T1 murine mammary carcinoma, a common animal model of metastatic breast cancer. A mildly raised temperature (i.e.39°C) was imposed on the skin surface of the implanted tumor using a thermal heating pad. Periodic heating (12 hours per day) was carried out for 3 days, 7 days, 14 days, and 21 days, respectively. The tumor growth rate was found significantly decreased in comparison to the control without hyperthermia. Biological evidences associated with tumor angiogenesis and metastasis were examined using histological analyses. Accordingly, the effect of mild hyperthermia on immune cell infiltration into tumors was also investigated. It was demonstrated that a delayed tumor growth and malignancy progression was achieved by mediating tumor cell apoptosis, vascular injury, degrading metastasis potential and as well as inhibiting the immunosuppressive cell myeloid derived suppressor cells (MDSCs) recruitment. Further mechanistic studies will be performed to explore the quantitative relationship between tumor progression and thermal dose in the near future.

Design of microprobe for accurate thermal treatment of tumor.

Thermal treatment has become an alternative modality for cancer treatment. Low temperature freezing and high temperature heating kill tumor cells effectively through direct and indirect injuries by biochemical and physical stresses. Hyperthermia at a mildly elevated temperature has also been reported to induce biochemical alternations to kill tumor cells and to stimulate immunological response to prevent metastasis. The comprehensive multi-scale biological responses to different thermal history experienced demand an accurate temperature control of the thermal system used for such a treatment. A thermal system was built in our lab utilizing RF heating and liquid nitrogen cooling through a needle probe. In practice, difficulties involved in temperature measurement for in vivo monitoring and control of thermal input through two-phase LN2 flow inside the probe compromise the treatment outcome. To ensure an accurate temperature control, a new model was developed to study the dynamic freezing capacity of the cryo-probe by accounting for the probe shape and dimensions. The model was validated by experiments and used to predict the freezing processes under different conditions. Numerical simulation results showed that combined with RF heating, the system could be used to perform different treatment protocols with an accurate temperature control.

Design of a new probe for tumor treatment in the alternate thermal system based on numerical simulation.

A new probe for tumor treatment is designed and simulated in this study. This probe combines the cryosurgery and hyperthermia which is suitable for the treatment of subcutaneous tumors. Simulations of the cooling and heating processes demonstrate that the probes are capable of treating the tumor effectively. And the numerical results indicate that the lengths of the probe, the diameters of the inner tube and the pressures of liquid nitrogen influence the probes' cooling ability. The temperature responses at the tumor base induced by different probes are similar, though the great differences appear on the treatment interface of the probes, thus the temperature gradient within the tumor. Based on the simulation results, the heating effect of the probe is shown to be effective in damaging the tumor while protecting normal tissue in the surrounding. Animal experiments will be carried out using this type of probe to treat tumor in the near future.

Immunologic response induced by synergistic effect of alternating cooling and heating of breast cancer.

In this study, the therapeutic effect and the induced anti-tumor immunity through the alternating cooling and heating was investigated using 4T1 murine mammary carcinoma, a common model of human metastatic breast cancer. While fifteen of seventeen regular mice were cured, primary recurrence and metastasis caused death of all the nude mice within one month after the same treatment. Histological analyses showed that viable cells existed in the tumor debris after the treatment, indicating that the direct killing effect was not the only therapeutic mechanism. Further investigation found rejection of tumor upon re-challenge, and anti-tumor immune response was studied. Stronger cytotoxicity T-lymphocyte (CTL) and Th1 cytokines response as well as infiltration of immunocytes were observed in the treated mice in comparison to those after the surgical resection. The results showed that the alternating cooling and heat could stimulate anti-tumor immunologic response in vivo and the underlying mechanisms will be further investigated in the near future.

Study on tumor microvasculature damage induced by alternate cooling and heating.

Tumor vasculature damage induced by various thermal treatments has been studied in vivo via laser confocal microscopy. Murine mammary carcinoma 4T1 was implanted in the nude mice dorsal skin fold window chamber. The implanted tumor was treated by alternate cooling and heating. Results showed that the treatment was much more effective as compared with that of cooling or heating alone, especially in damaging the tumor vasculature. In general, tumor vascular response to thermal stimuli was heterogeneous. All the treatments of hyperthermia at 42 degrees C (for 1 h), alternate cooling at 1 degrees C and heating at 42 degrees C (for 1/2 h each) and that of -10 degrees C/42 degrees C (for 1/2 h each) enhanced liposome extravasation. Pre-cooling tumor at 1 degrees C preserved most of the vascular integrity but partially inhibited the effect of post-hyperthermia at 42 degrees C. On the other hand, cooling at -10 degrees C for 1/2 h before heating at 42 degrees C caused severe vessel damage. Histo-pathological analyses further confirmed the effect as rare tumor vessel recurrence and large necrotic tumor tissue areas shown on the 7th day after the treatment.

Immunological response induced by alternated cooling and heating of breast tumor.

A new in-situ thermal physical method combining both cryosurgery and local hyperthermia was used to treat mice bearing 4T1 murine mammary carcinoma. The induced anti-tumor immune response was investigated. The cryo/heat treatment resulted in stimulation of CTL response and attraction of immunocytes into the tumor debris, which correlated well to the tumor rejection in re-implantation. The results suggested that alternated cooling and heating had synergistic effect and might be developed into an alternative modality for tumor therapy.

Enhanced efficacy of anti-tumor liposomal doxorubicin by hyperthermia.

The efficiency of novel tumor chemotherapeutics could be increased using targeted drug delivery by hyperthermia. In this paper, the 3D liposomal doxorubicin distribution in the tumor tissue enhanced by local hyperthermia was quantitatively studied in real time using laser confocal microscopy. Results showed that the thermally induced liposomal doxorubicin extravasation was non-uniform and more excessive in the peripheral region than that in the tumor center. The effect of the thermally targeted drug delivery was also investigated. On the 1st, 3rd day after the thermally targeted drug treatment, histological examination showed that many nucleolus were condensed and collapsed in the peripheral region. But, in the tumor center, there were no such changes found until the 3rd day. While on the 6th day, tumor cells in both the peripheral and center region were found necrotic. The enhancement of the nanoparticle anti-tumor drug effect was significant. A theoretical analysis of liposomal doxorubicin diffusion to the tumor cells in vivo was performed. Results showed that it took more than 40 hrs for the doxorubicin to get into the tumor cells in the center region from the periphery region. The theoretical results well explained the experimental observations.

Simultaneous measurements of local tissue temperature and blood perfusion rate in the canine prostate during radio frequency thermal therapy.

Local tissue temperature and blood perfusion rate were measured simultaneously to study thermoregulation in the canine prostate during transurethral radio-frequency (RF) thermal therapy. Thermistor bead microprobes measured interstitial temperatures and a thermal clearance method measured the prostatic blood perfusion rate under both normal and hyperthermic conditions. Increase in local tissue temperature induced by the RF heating increased blood perfusion throughout the entirety of most prostates. The onset of the initial increase in blood perfusion was sometimes triggered by a temporal temperature gradient at low tissue temperatures. When tissue temperature was higher than 41 degrees C, however, the magnitude and the spatial gradient of temperature may play significant roles. It was found that the temperature elevation in response to the RF heating was closely coupled with local blood flow. The resulting decrease in or stabilization of tissue temperature suggested that blood flow might act as a negative feedback of tissue temperature in a closed control system. Results from this experiment provide insights into the regulation of local perfusion under hyperthermia. The information is important for accurate predictions of temperature during transurethral RF thermal therapy.

Tissue temperature oscillations in an isolated pig kidney during surface heating.

In the present study, an isolated pig kidney was used to study tissue temperature oscillations due to vascular thermoregulation, frequently observed during hyperthermia treatments. The kidney was perfused with the distilled water pumped through the renal artery to simulate blood flow. When the local perfusion rate was increased with a time delay, temperature oscillations were observed in the kidney as its surface temperature raised linearly with time in a water bath. The magnitude of tissue temperature decreased as the flow rate increased during the surface heating. A 3D transient model was developed to predict the temperature oscillations, which was validated by the measurements. Using the model, relationships of the changes in perfusion rate and heating rate with temperature oscillations were investigated. It was found that the heating rate, and the magnitude and time delay of the flow response to the temperature elevation, each significantly affected tissue temperature oscillations. The magnitude of oscillation was primarily determined by the spatial gradient of temperature, while the oscillation type depended on the change of flow rate and the time delay. In conclusion, to accurately predict and control the tissue temperature distribution during hyperthermia treatment, understanding of the local perfusion change with respect to tissue temperature is essential.