Distinct Tumor Microenvironment at Tumor Edge as a Result of Astrocyte Activation Is Associated With Therapeutic Resistance for Brain Tumor

Tumor vasculatures and hypoxia are critical tumor micro-environmental factors associated with tumor response to the therapy and heterogeneous in both time- and location-dependent manner. Using a murine orthotopic anaplastic astrocytoma model, ALTS1C1, this study showed that brain tumor edge had a very unique microenvironment, having higher microvascular density (MVD) and better vessel function than the tumor core, but on the other hand was also positive for hypoxia markers, such as pimonidazole (PIMO), hypoxia inducible factor-1α (HIF-1α), and carbonic anhydrase IV (CAIX). The hypoxia at tumor edge was transient, named as peripheral hypoxia, and caused by different mechanisms from the chronic hypoxia in tumor core. The correlation of CAIX staining with astrocyte activation marker, glial fibrillary acid protein (GFAP), at the tumor edge indicated the involvement of astrocyte activation on the development of peripheral hypoxia. Peripheral hypoxia was a specific trait of orthotopic brain tumors at tumor edge, regardless of tumor origin. The hypoxic cells were resistant to the therapy, regardless of their location. Surviving cells, particularly those at the hypoxic region of tumor edge, are likely the cause of tumor recurrence after the therapy. New therapeutic platform that targets cells in tumor edge is likely to achieve better treatment outcomes

Repeated Small Perturbation Approach Reveals Transcriptomic Steady States

The study of biological systems dynamics requires elucidation of the transitions of steady states. A “small perturbation” approach can provide important information on the “steady state” of a biological system. In our experiments, small perturbations were generated by applying a series of repeating small doses of ultraviolet radiation to a human keratinocyte cell line, HaCaT. The biological response was assessed by monitoring the gene expression profiles using cDNA microarrays. Repeated small doses (10 J/m2) of ultraviolet B (UVB) exposure modulated the expression profiles of two groups of genes in opposite directions. The genes that were up-regulated have functions mainly associated with anti-proliferation/anti-mitogenesis/apoptosis, and the genes that were down-regulated were mainly related to proliferation/mitogenesis/anti-apoptosis. For both groups of genes, repetition of the small doses of UVB caused an immediate response followed by relaxation between successive small perturbations. This cyclic pattern was suppressed when large doses (233 or 582.5 J/m2) of UVB were applied. Our method and results contribute to a foundation for computational systems biology, which implicitly uses the concept of steady state

Distinct Role of CD11b+Ly6G−Ly6C− Myeloid-Derived Cells on the Progression of the Primary Tumor and Therapy-Associated Recurrent Brain Tumor

Myeloid-derived cells have been implicated as playing essential roles in cancer therapy, particularly in cancer immunotherapy. Most studies have focused on either CD11b+Ly6G+Ly6C+ granulocytic or polymorphonuclear myeloid-derived suppressor cells (G-MDSCs or PMN-MDSCs) or CD11b+Ly6G−Ly6C+ monocytic MDSCs (M-MDSCs), for which clear roles have been established. On the other hand, CD11b+Ly6G−Ly6C− myeloid-derived cells (MDCs) have been less well studied. Here, the CD11b-diphtheria toxin receptor (CD11b-DTR) transgenic mouse model was used to evaluate the role of CD11b+ myeloid-derived cells in chemotherapy for an orthotopic murine astrocytoma, ALTS1C1. Using this transgenic mouse model, two injections of diphtheria toxin (DT) could effectively deplete CD11b+Ly6G−Ly6C− MDCs while leaving CD11b+Ly6G+Ly6C+ PMN-MDSCs and CD11b+Ly6G−Ly6C+ M-MDSCs intact. Depletion of CD11b+Ly6G−Ly6C− MDCs in mice bearing ALTS1C1-tk tumors and receiving ganciclovir (GCV) prolonged the mean survival time for mice from 30.7 to 37.8 days, but not the controls, while the effectiveness of temozolomide was enhanced. Mechanistically, depletion of CD11b+Ly6G−Ly6C− MDCs blunted therapy-induced increases in tumor-associated macrophages (TAMs) and compromised therapy-elicited angiogenesis. Collectively, our findings suggest that CD11b+Ly6G−Ly6C− MDCs could be manipulated to enhance the efficacy of chemotherapy for brain tumors. However, our study also cautions that the timing of any MDC manipulation may be critical to achieve the best therapeutic result

Radiation therapy-induced tumor invasiveness is associated with SDF-1-regulated macrophage mobilization and vasculogenesis.

Radiation therapy (RT) remains the front-line treatment for high-grade gliomas; however, tumor recurrence remains the main obstacle for the clinical success of RT. Using a murine astrocytoma tumor cell line, ALTS1C1, the present study demonstrates that whole brain irradiation prolonged the survival of tumor-bearing mice, although the mice eventually died associated with increased tumor infiltration. Immunohistochemical (IHC) analysis indicated that RT decreased the microvascular density (MVD) of the primary tumor core, but increased the MVD of the tumor invasion front. RT also increased the number of tumor-associated macrophages (TAMs) and the expression of stromal cell-derived factor-1 (SDF-1) and hypoxia-inducible factor-1 (HIF-1) at the tumor invasion front. SDF-1 expression suppressed by siRNA (SDFkd tumors) showed a decrease in RT-enhanced tumor invasiveness, leading to prolonged survival of mice bearing these tumors. The invasion front in SDFkd tumors showed a lower MVD and TAM density than that in the islands of the control or irradiated ALTS1C1 tumors. Our results indicate that tumor-secreted SDF-1 is one key factor in RT-induced tumor invasiveness, and that it exerts its effect likely through macrophage mobilization and tumor revascularization

The Roles of Macrophages and Nitric Oxide in Interleukin-3-Enhanced HSV-Sr39tk-Mediated Prodrug Therapy

<div><p>The herpes simplex virus thymidine kinase/ganciclovir (HSV-sr39tk/GCV) system is a well-established prodrug system used in cancer gene therapy. However, this system is currently not effective enough to eradicate malignant tumors completely. This study aimed to evaluate whether co-expression of interleukin-3 (IL-3) could enhance the anti-tumor activity of HSV-sr39tk/GCV prodrug gene therapy using a murine TRAMP-C1 prostate tumor model. <i>In vitro</i> results demonstrated that HSV-sr39tk-transfected cells exhibited enhanced sensitivity to the GCV prodrug, which was not affected by co-expression of the mIL-3 gene. However, <i>in vivo</i> studies showed that co-expression of the mIL-3 gene significantly increased the HSV-sr39tk/GCV-induced tumor growth delay and even cured the tumor. The TRAMP-C1-specific immune response of spleen lymphocytes from mice bearing HSV-sr39tk- and IL-3-expressing TRAMP-C1 tumors was measured by ELISA. Results showed that IL-3-activated IL-4-dominant lymphocytes became IFN-γ- dominant lymphocytes after combined HSV-sr39tk/GCV therapy. The efficacy of combined therapies on tumor regression was reduced when macrophages populations were depleted by carrageenan or NO production was inhibited by administration of the iNOS inhibitor, L-NAME. These results suggest that utilizing a bicistronic vector to express HSV-sr39tk and the IL-3 gene induced an enhanced macrophage- or NO-dependent anti-tumor effect.</p> </div

The role of macrophages in IL-3-enhanced HSV-tk/GCV therapy.

<p>(A) The percentage of CD11b⁺, CD68⁺, and Gr-1⁺ cells in various gene-transfected tumors was determined by flow cytometry. When a diameter of 6 mm was reached, the tumors were digested to produce a single cell suspension. Tumor-infiltrating cells were studied with flow cytometry. Data and error bars are means ± SDs for n = 3 mice per group. (B) The production of IFN-γ by 2×10⁵ peritoneal macrophages after co-culture with 2×10⁵ transfected cells and treatment with ganciclovir (GCV) for three days. The concentration of IFN-γ in the media was measured by ELISA. Data and error bars are means ± SDs of three independent experiments. (C) Representative flow cytometry profiles to show the changes of splenocyte population after administration of carrageenan. Mice were injected i.p. with carrageenan to deplete macrophages at 7, 3, and 1 day prior to sacrifice. R: red blood cells, L: lymphocytes, M: monocytes, G: granulocytes. (D) The <i>in vivo</i> tumor growth curves of various gene-transfected TRAMP-C1 tumors following GCV treatment in carrageenan-treated mice. Mice were injected i.p. with carrageenan to deplete macrophages at 7, 3, and 1 day prior to tumor inoculation, and this continued twice per week after tumor implantation until mice were sacrificed. GCV was injected twice per day for five continuous days when tumors reached 5 mm in diameter. Tumor sizes were measured every two days. Data and error bars are means ± SDs for at least three mice per group. *indicates <i>P</i><0.05, **indicates <i>P</i><0.01, ***indicates <i>P</i><0.001 determined by the one way ANOVA.</p

Relative ratio of tumor diameters after GCV/carrageenan administration or L-NAME administration.

<p>The diameter of tumors without GCV administration was compared at day nine with the initial diameter (5 mm) at day 0. The diameter of tumors from mice received GCV treatment was compared at day 23 with the initial diameter (5 mm) at day 0 before GCV treatment.</p>*<p>indicates <i>P</i><0.05 when the TRAMP-C1/sr39tk and TRAMP-C1/mIL3-sr39tk groups were compared.</p>#<p>indicates <i>P</i><0.05 when the carrageenan, L-NAME and SCID groups were compared with control group. <i>P</i> was determined by one way ANOVA test.</p

The <i>in vivo</i> responses of various gene-transfected TRAMP-C1 tumors in L-NAME-treated mice to GCV treatment.

<p>When tumor diameters reached 5 mm, mice were injected i.p. with GCV for five consecutive days and received L-NAME in drinking water for the remaining experimental period. Tumor volumes were measured every two days. Data and error bars are means ± SDs for at least three mice per group. **indicates <i>P</i><0.01 determined by the Student’s t-test.</p

The effect of iNOS inhibition on HSV-sr39tk/GCV and IL-3 therapies.

<p>(A) Gene expression of CD11b⁺ cells isolated from tumors as analyzed by RT-PCR. When tumor diameters reached 6–8 mm, tumors were resected and digested to produce a single cell suspension. CD11b⁺ cells were isolated using a MACS CD11b MicroBeads cell separation kit. Total RNA was extracted, reverse transcribed, and amplified with specific primers for 30 cycles. (B) Representative images of iNOS expression from mice in each group. iNOS gene expression was analyzed by immunohistochemical staining. Tumor tissue was stained using anti-iNOS (green) and anti-CD11b/CD68 (red) antibodies. White arrows indicate iNOS⁺/CD11b⁺ or CD68⁺ cells and white stars indicate iNOS⁺/CD11b⁻ or CD68⁻ cells. Original magnification: ×400. Scale bar: 50 µm. Images are representative results from individual animals in each group. (C) Percentages of CD11b⁺iNOS⁺ and CD11b⁻iNOS⁺ were quantified by Image-Pro Plus 6.0 software. *indicates <i>P</i><0.05 determined by the Student’s t-test.</p