The ability to modulate myofibroblast phenotype will have an impact in wound healing, aging, cancer, and Dupuytren’s contracture development. Our project focused on investigating the effect of 808 nm laser wavelength on the presence of myofibroblast and contraction using anchored delayed release collagen lattice models. Fibroblast cells were cultured and suspended in collagen lattices in the presence or absence of glycated chitosan (GC) to mimic a wound healing environment in-vitro. The cells were allowed to generate maximum tension for 6 days followed by laser light stimulation. The total number of cells and presence of fibroblast proliferation and differentiation into myofibroblasts was determined using immunostaining followed by fluorescent microscope photography. Lattice tension generation was assessed by releasing the lattices from their anchorage and measuring their diameters. Control lattices contracted more than GC lattices. Myofibroblast differentiation was slightly higher in the control group. Repeats of the experiment are needed to confirm our results to determine if near infra-red laser radiation can be applied to treat open wounds and study tumor stromas.
Non-invasive laser immunotherapy (NLIT) is a viable alternative to traditional cancer treatment because it combines the photothermal and immunological effects of non-invasive laser irradiation and single-walled carbon nanotubes (SWNT) with an immunoadjuvant, glycated chitosan (GC). This combination forms SWNT-GC, a photosensitive immunoadjuvant, which creates a tumor-specific immunity that targets both the primary tumor and any metastasis. It is known that NLIT induces anti-tumor as well as anti-metastatic immune responses, but its immunological mechanism is not clear. The objective of this study is to clarify the role of SWNT-GC in cancer cell migration. Panc02 (non-metastatic) and Panc02-H7 (metastatic) pancreatic cancer cells were used in two-dimensional elastomer plug assays to observe the restriction of cell migration induced by SWNT, GC, and SWNT-GC individually. To replicate a three-dimensional in vivo study, a similar assay was repeated using embedded collagen lattices. Both the 2D and the 3D studies confirmed previous results indicating that GC inhibits cancer cell motility. The 2D and 3D studies also showed that SWNT-GC inhibited the migration of cancer cells, but a discrepancy was observed regarding the effect of SWNT alone. The 2D model concluded that SWNT inhibited migration while the 3D model determined that SWNT promoted migration. The results of this study will guide future work to determine the mechanism behind NLIT, including how metastases are eradicated and how the tumor specific immunity is created.
KEYWORDS: Laser irradiation, Cancer, Single walled carbon nanotubes, In vitro testing, Resistance, Optical properties, In vivo imaging, Tumors, Biomedical optics, Medical research, Biology
Laser immunotherapy (LIT) uses laser irradiation and immunological stimulation to target all types of metastases and creates a long-term tumor resistance. Glycated chitosan (GC) is the immunological stimulant used in LIT. Interestingly, GC can act as a surfactant for single-walled carbon nanotubes (SWNTs) to immunologically modify SWNTs. SWNT-GC retains the optical properties of SWNTs and the immunological functions of GC to help increase the selectivity of the laser and create a more optimal immune response. One essential aspect of understanding this immune response is knowing how laser irradiation affects cancer cells’ ability to metastasize. In this experiment, a cell migration assay was performed. A 2mm circular elastomer plugs were placed at the bottom of multi-well dishes. Pre-cancerous keratinocytes, different tumor cells, and fibroblasts were then plated separately in treated wells. Once the cells reached 100% confluence, they were irradiated by either a 980nm or 805nm wavelength laser. The goal was to determine the effects of laser irradiation and immunological stimulation on cancer cell migration in vitro, paying the way to understand the mechanism of LIT in treating metastatic tumors in cancer patients.
The microenvironments of tumors are involved in a complex and reciprocal dialog with surrounding cancer cells. Any novel treatment must consider the impact of the therapy on the microenvironment. Recently, clinical trials with laser immunotherapy (LIT) have proven to effectively treat patients with late-stage, metastatic breast cancer and melanoma. LIT is the synergistic combination of phototherapy (laser irradiation) and immunological stimulation. One prominent cell type found in the tumor stroma is the fibroblast. Fibroblast cells can secrete different growth factors and extracellular matrix modifying molecules. Furthermore, fibroblast cells found in the tumor stroma often express alpha smooth muscle actin. These particular fibroblasts are coined cancer-associated fibroblast cells (CAFs). CAFs are known to facilitate the malignant progression of tumors. A collagen lattice assay with human fibroblast cells is used to elucidate the effects LIT has on the microenvironment of tumors. Changes in the contraction of the lattice, the differentiation of the fibroblast cells, as well as the proliferation of the fibroblast cells will be determined.
Combining near infrared (NIR) laser irradiation into a tumor treatment therapy has shown promising results. For a comprehensive tumor therapy, it is important to understand the effects of NIR irradiation not only on the tumor, but on the tumor stroma as well. The composition of the microenvironment present near the tumor cells is critical to the phenotype of the tumor. Fibroblasts affect tissue homeostasis and change the microenvironment surrounding the tumor. Myofibroblast are derived from fibroblast cells, and in some cases indicate the transformation of healthy tissue into malignant tissue. Wound healing environments are rich in fibroblast cells and are similar to tumor stromas. To simulate a tumor stroma a wound healing environment was constructed. Two different human fibroblast cells were cultured in collagen lattices. Specifically, collagen lattices were created, with type 1 collagen, incubated for 5 days and irradiated with a 980nm laser on the 4th day. The subsequent collagen lattices were either released and measured, or fixed for immunostaining on the 5th day; the contraction rates also were analyzed. Furthermore, collagen lattices were stained to identify fibroblast proliferation and differentiation, into myofibroblasts. The results suggested NIR laser irradiation had some biological effects on the fibroblast cells, but the full extent of the effects is still unclear.
The ability to modulate the myofibroblast phenotype will have important implications in wound healing, aging and
cancer development. Our objective was to determine whether irradiation using a 980-nm laser affects the presence of
myofibroblasts and cellular contractility using an attached collagen lattice model. Fibroblasts in type I collagen lattices
were allowed to generate tension for 5 days in the presence or absence of laser light stimulation on day 4.
Immunostaining was used to determine the total number of cells in a representative image and percentage of
myofibroblasts in the same image; tension generation was determined by releasing tension and measuring diameter
change over time. One treatment demonstrated a slight lattice contraction increase over control, correlated with increased
cell number. Myofibroblast percentage was low and was not correlated with lattice contraction. More experiments are
necessary due to limited sample size and lack of large deviations from the control.
Laser immunotherapy (LIT) has shown great promise in pre-clinical studies and preliminary clinical trials.
It could not only eradicate treated local tumors but also cause regression and elimination of untreated
metastases at distant sites. Combining a selective photothermal therapy with an active immunological
stimulation, LIT can induce systemic anti-tumor immune responses. Imiquimod (IMQ), a toll-like receptor
agonist, was used for the treatment of late-stage melanoma patients and glycated chitosan (GC), a biological
immunological modulator, was used for the treatment of late-stage breast cancer patients, in combination of
irradiation of a near-infrared laser light. To observe the immunological changes before and after LIT
treatment, the pathological tissues of melanoma and breast cancer patients were processed for
immunohistochemical analysis. Our results show that LIT changed the expressions of several crucial T cell
types. Specifically, we observed significant decreases of CD3+
T-cells and a significant increase of CD4+,CD8+, and CD68+ T-cells in the tumor samples after LIT treatment. While not conclusive, our study could
shed light on one the possible mechanisms of anti-tumor immune responses induced by LIT. Further
studies will be conducted to identify immunological biomarkers associated with LIT-induced clinical
response.
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