<p>Drug resistance in cancer. (<b>A</b>) Matrix changes in cancer [<a href="#B39-targets-02-00015" class="html-bibr">39</a>]; (<b>B</b>) schematic representation of (A) neurovascular unit; (B) paracellular transport pathway and transcellular transport pathway of BBB; (C) tight junction (TJ) associated components [<a href="#B62-targets-02-00015" class="html-bibr">62</a>]. (Figure was created by <a href="http://biorender.com" target="_blank">biorender.com</a> accessed on 30 August 2024).</p> Full article ">Figure 2
<p>Management of cancer evolution and early intervention. (<b>A</b>) The heterogeneity of non-silent mutations from multiple-sample sequencing across a range of cancer types (black circles represent treatment naive tumors, with red triangles indicating tumors that have received treatment) [<a href="#B9-targets-02-00015" class="html-bibr">9</a>]; (<b>B</b>) evolutionary trees illustrating intratumor heterogeneity across cancer types [<a href="#B9-targets-02-00015" class="html-bibr">9</a>]; (<b>C</b>) phylogenetic relationships of the tumor regions [<a href="#B68-targets-02-00015" class="html-bibr">68</a>]; (<b>D</b>) clinical applications of CTC and ctDNA analyses in cancer care [<a href="#B72-targets-02-00015" class="html-bibr">72</a>]. (<b>E</b>) ctDNA-based MRD testing is predictive of survival outcomes in postsurgical patients with colorectal cancer [<a href="#B73-targets-02-00015" class="html-bibr">73</a>]. (<b>F</b>) A BEAMing analysis of circulating tumor DNA of patients with acquired resistance to cetuximab or panitumumab displays complex patterns of KRAS and NRAS mutations [<a href="#B74-targets-02-00015" class="html-bibr">74</a>]. (<b>G</b>) Resistance to EGFR therapy is reversed by pharmacological inhibition of EGFR and MEK (a combinatorial treatment with cetuximab plus pimasertib is effective in inducing tumor shrinkage in vivo) [<a href="#B74-targets-02-00015" class="html-bibr">74</a>]. (<b>H</b>) The evolution of resistance in a metastatic lesion. As the lesion (green) grows from one cell to a detectable size, new resistant subclones appear. Some of them are lost to stochastic drift (yellow and pink), while others survive (purple, red and orange triangles). Instead of looking at the time of appearance of new clones, the approach takes into account the total size of the lesion when the resistance mutation first occurred [<a href="#B75-targets-02-00015" class="html-bibr">75</a>]. (<b>I</b>) Resistant subclones in metastatic lesions [<a href="#B75-targets-02-00015" class="html-bibr">75</a>]. (<b>J</b>) The number of circulating tumor DNA (ctDNA) fragments per milliliter (Y1 to Y4) harboring different mutations associated with resistance to anti-EGFR agents in colorectal cancer patients treated with an EGFR blockade. The ratio of resistant clone sizes is given by the ratio of the ctDNA counts for any two resistance-associated mutations [<a href="#B75-targets-02-00015" class="html-bibr">75</a>]. (<b>K</b>) Heterologous vaccination with ChAd68 and samRNA induces broad, durable CD8<sup>+</sup> T cell responses in NHPs that are detectable long-term and can be boosted ≥ 2 years after their prime [<a href="#B76-targets-02-00015" class="html-bibr">76</a>]. (<b>L</b>) The removal of immunodominant epitopes and repetition of epitopes leads to increased target density and T cell response to KRAS mutant neoantigens [<a href="#B77-targets-02-00015" class="html-bibr">77</a>].</p> Full article ">Figure 3
<p>Employing vascular endothelial growth factor inhibitors. (<b>A</b>) A summary of some of the major molecules implicated in angiogenesis [<a href="#B128-targets-02-00015" class="html-bibr">128</a>]. (<b>B</b>) Potential mechanisms of resistance to targeted VEGF therapy in cancer [<a href="#B128-targets-02-00015" class="html-bibr">128</a>]. In established tumours, VEGF blockade aggravates hypoxia, which upregulates the production of other angiogenic factors or increases tumour cell invasiveness (a). Other modes of tumour vascularisation, including intussusception, vasculogenic mimicry, differentiation of putative cancer stem cells into endothelial cells, vasculogenic vessel growth, and vessel co-option, might be less sensitive to VEGF blockade (b). Tumour vessels covered by pericytes are less sensitive to VEGF blockade (c). Recruited proangiogenic bone-marrow-derived cells, macrophages or activated cancer-associated fibroblasts can rescue tumour vascularisation by production of proangiogenic factors (d). (<b>C</b>) A plot of the Kaplan–Meier estimates for progression-free survival (non-small-cell lung cancer) for the (a) 7.5 mg/kg bevacizumab (Bev) arm and the (b) 15 mg/kg bevacizumab arm compared with a placebo [<a href="#B122-targets-02-00015" class="html-bibr">122</a>]. (<b>D</b>) The effect of a single injection of bevacizumab on the structural and functional markers of vascular normalization [<a href="#B123-targets-02-00015" class="html-bibr">123</a>]. (a) Microvessel density decreased. (b) Bevacizumab did not affect the density of mature vessels. (c) Fraction of vessel perimeter associated with pericytes (αSMA+ cells), a marker that distinguishes between poorly and completely covered vessels, increased. (d) Interstitial fluid pressure, which is a functional measurement of vessel leakiness and lymphatic vessel dysfunction, decreased. (e,f) Histological markers of functional vascular normalization, Ki67 for proliferation and HIF-1α, did not change significantly. In order to distinguish the figure and subfigure, the subfigures’ labels are replaced by lowercase letters in annotation.</p> Full article ">Figure 4
<p>Enhancing pericyte coverage. (<b>A</b>) Neural cell adhesion molecule deficiency induces increased tumor blood vessel leakage during β tumor cell progression [<a href="#B129-targets-02-00015" class="html-bibr">129</a>]. NCAM deficiency induces increased Pancreas sections from 8-week-old RT (a) and RT<sup>NCAM+/−</sup> mice (b) were stained with H&E. (b) Isolated cell clusters were specifically found inside blood-filled cavities within RT<sup>NC/KO</sup> islets (arrow). Inset in b shows a higher magnification of the isolated cell cluster. (c–h) Pancreas sections of mice perfused with FITC-dextran (green) were double-immunostained with antibodies against PECAM (red) and insulin (blue). (c,d) Islets from WT (c) and NCAM<sup>−/−</sup>(d) mice. (e–h) Angiogenic islets from 8-week-old RT (e,g) and RT<sup>NCAM+/−</sup>(f,h) mice. The islet area is indicated by dashed lines, and extravascular and intravascular FITC-dextran is indicated by arrowheads and arrows, respectively, in inset in (e). Dashed lines in g and h mark blood-filled cavities. FITC-dextran specifically leaked into RT<sup>NC/KO</sup> blood-filled cavities (h). (i) The percentage of islets containing blood-filled cavities was higher in RT<sup>NC/KO</sup> compared with RT mice at 8 weeks of age (χ<sup>2</sup> test, *** <span class="html-italic">p</span> < 0.001). (j) Distribution of RT and RT<sup>NC/KO</sup> islets at 8 weeks of age according to their vessel leakage (grades 0–3, where grade 3 includes islets with most extensive leakage). (<b>B</b>) The pathological organization of periendothelial α-SMA<sup>+</sup> cells correlates with increased tumor vessel leakage in RT<sup>NC/KO</sup> angiogenic islets [<a href="#B129-targets-02-00015" class="html-bibr">129</a>]. Pancreas sections from 8-week-old mice were double-immunostained with antibodies against PECAM (red) and α-SMA (a–e, green; f, red). In WT (a) and NCAM<sup>−/−</sup> (b) islets, α-SMA<sup>+</sup> cells were closely attached to the endothelium. Premature abnormal organization of periendothelial α-SMA<sup>+</sup> cells, including detachment of α-SMA<sup>+</sup> cells from endothelial cells (arrow in c) and multiple layers of α-SMA<sup>+</sup> cells with an apparent loose attachment to the endothelium (d), and presence of fibroblastlike α-SMA<sup>+</sup> cells in RT<sup>NCAM+/−</sup> angiogenic islets (e), were observed in RT<sup>NCAM+/−</sup> islets. The dashed lines and arrows in d indicate the borders of the endothelium and α-SMA<sup>+</sup> cells stretching away from the vessel, respectively. (f) Pancreas section of an 8-week-old RT<sup>NCAM+/−</sup> mouse perfused with FITC-dextran (green), immunostained with anti–α-SMA (red). Increased leakage correlated with severely disorganized α-SMA<sup>+</sup> periendothelial cells. The inset in (c) shows coexpression of NG2 (green) and α-SMA (red). (g) Analysis of blood vessel density revealed no difference between RT and RT<sup>NC/KO</sup> islets. (<b>C</b>) The effect of host-derived Ang-2 deficiency on MVD (a,b), the diameter of intratumoral microvessels (a,b), and perfusion (c) in Lewis lung carcinoma tumors [<a href="#B130-targets-02-00015" class="html-bibr">130</a>]. (<b>D</b>) Structural changes within established primary tumor vessels invoked by vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibition [<a href="#B134-targets-02-00015" class="html-bibr">134</a>]. (a) Colocalization of CD31-positive area (endothelial cells) and NG2-positive area (perivascular cells) (* <span class="html-italic">p</span> = 0.05 by two-sided t test; n = 6 per group) in 4T1 tumors. (b) Colocalization of CD31-positive area (endothelial cells) and desmin-positive area (perivascular cells) (* <span class="html-italic">p</span> = 0.01 by two-sided t test; n = 6 per group) in 4T1 tumors. (c) Mean distance between desmin-positive pericytes and CD31 = positive endothelial cells in microns (* <span class="html-italic">p</span> < 0.01 by two-sided t test; n = 6 per group) in 4T1 tumors. (d) Colocalization of CD31+ area (endothelial cells) and NG2+ area (perivascular cells) (* <span class="html-italic">p</span> = 0.05 by two-sided t test; n = 6 per group) in E0771 tumors (* <span class="html-italic">p</span> < 0.01 by two-sided t test; n = 6 per group). (e) Enhanced vascular pericyte coverage in AKB-9778–treated tumors (right panel) compared with control-treated tumors (left panel) (red: CD31; green: NG2; blue: DAPI). (f,g) Vessel diameter in control vs. AKB-9778–treated 4T1 (f) and E0771 (g) tumors (f: * <span class="html-italic">p</span> < 0.01 by two-sided t test, n = 6 per group; g: * <span class="html-italic">p</span> < 0.01 by two-sided t test, n = 6 per group). (h,i) Vessel density in control vs AKB-9778–treated 4T1 (h) and E0771 (i) tumors (h: * <span class="html-italic">p</span> = 0.05 by two-sided t test, n = 6 per group; i: * <span class="html-italic">p</span> = 0.05 by two-sided t test, n = 6 per group). (<b>E</b>) Ang2-blocking antibodies inhibit primary tumor growth, angiogenesis, and lymphangiogenesis [<a href="#B132-targets-02-00015" class="html-bibr">132</a>]. (a) Growth curves of LNM35 primary tumors in nu/nu mice treated with the Ang2-blocking antibodies or hIgG control, n = 8 in both groups. (b) Tumor weights at excision 16 days after implantation, <span class="html-italic">p</span> = 0.002. Student’s <span class="html-italic">t</span> test. (c) Representative immunohistochemical images of LYVE-1- and CD31-stained tumor sections. (d) Quantification of densities and area fractions of Lyve-1-positive lymphatic vessels and of CD31-positive blood vessels from at least five histological sections, <span class="html-italic">p</span> = 0.013 and 0.019, respectively. Student’s <span class="html-italic">t</span> test. (<b>F</b>) The Ang2-blocking antibody induces internalization of the Ang2-Tie2 complexes, leaving Ang1-Tie2 complexes intact at endothelial cell–cell junctions [<a href="#B132-targets-02-00015" class="html-bibr">132</a>]. (<b>G</b>) The overall survival in patients with ascites at baseline (trebananib plus weekly paclitaxel in recurrent ovarian cancer) [<a href="#B133-targets-02-00015" class="html-bibr">133</a>]. In order to distinguish the figure and subfigure, the subfigures’ labels are replaced by lowercase letters in annotation.</p> Full article ">Figure 5
<p>Reinforcing tight junctions. (<b>A</b>) CU06-1004 reduces vascular leakage with a concomitant increase in junction integrity in tumor blood vessels [<a href="#B137-targets-02-00015" class="html-bibr">137</a>]. (a) Schematic plan for the administration of Sac-1004 or control (DMSO) to tumor-bearing mice. (b) B16F10 tumor-bearing mice (n = 5) were injected with Sac-1004 or control as in (a) and tumor vascular leakage was quantified by the Evans blue method. (c) Vascular leakage was assessed by FITC-dextran. (d) Images shown in (c) were quantified using ImageJ software. (e) Immunofluorescence staining of B16F10 tumor sections, treated with Sac-1004 or control, for CD31 and VE-cadherin. Arrows indicate discontinuity in VE-cadherin staining. (f) Quantification of immunofluorescence images shown in (e) using Multi Gauge software (n = 5). (g) LLC tumor sections, treated with Sac-1004 or control were costained for CD31, ZO-1 and DAPI. (h) Images shown in (g) were quantified using ImageJ software (n = 5). (i) Western blot analysis of B16F10 tumors treated with Sac-1004 or control for VE-cadherin. (j) VE-cadherin and actin blots from (i) were quantified using ImageJ software. * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001 (Student’s <span class="html-italic">t</span>-test). (<b>B</b>) CU06-1004 improves vascular perfusion, alleviates hypoxia, and normalizes tumor blood vessels in tumors [<a href="#B137-targets-02-00015" class="html-bibr">137</a>]. (a) Immunofluorescence staining of B16F10 tumor sections (n = 5), treated with Sac-1004 or control, for CD31 and tomato lectin. (b) Images shown in (a) were quantified using Image J software. (c) Immunohistochemical analysis of B16F10 tumor sections (n = 5) for CD31, hypoxia, and vascular perfusion (Hoechst dye) in the peritumoral and intratumoral zone. Arrows indicate non-perfused vessels. (d#x2013;f) Quantification of immunofluorescence images shown in (c) with Multi Gauge software. (g) Quantification of HIF-1α positive area using Multi Gauge software. (h) B16F10 tumor sections (n = 5), treated with Sac-1004 or control, were stained for CD31 and ColIV (up)/laminin (bottom). Arrowheads indicate the point of detachment between basement membrane and endothelial cells. (i) Quantification of basement membrane thickness in B16F10 tumor vessels shown in (h) using Multi Gauge software. (j) Immunofluorescence staining of LLC tumor sections (n = 5) for CD31 and NG2. Quantification was done using Multi Gauge software. * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001 (Student’s <span class="html-italic">t</span>-test). In order to distinguish the figure and subfigure, the subfigures’ labels are replaced by lowercase letters in annotation.</p> Full article ">Figure 6
<p>Normalizing intratumoral lymphatics while inhibiting peripheral tumor lymphangiogenesis. (<b>A</b>) The inhibition of tumor lymphangiogenesis by VEGFR−3−Ig (blood and lymphatic vessels in the LNM35 and N15 tumors) [<a href="#B139-targets-02-00015" class="html-bibr">139</a>]. Immunohistochemical staining is shown for LYVE-1 (a–d,g,h) to identify lymphatic vessels and for PECAM-1 (e,f) to identify both blood and lymphatic vessels. (a,c,e) Sections of the control LNM35/pEBS7 tumors. (b,d,f) Sections from the LNM35/vascular endothelial growth factor receptor−3−Ig (VEGFR−3−Ig) tumors. (g,h) Sections of the N15/pEBS7 and N15/VEGF−C tumors, respectively. (i) LYVE-1-stained or PECAM−1−-stained vessels in three microscopic fields of the highest vessel density were counted, and the results were compared by use of the unpaired t test. (<b>B</b>) The suppression of axillary lymph node metastasis by VEGFR−3−Ig [<a href="#B139-targets-02-00015" class="html-bibr">139</a>]. (a) Typical lymph nodes in mice bearing the vascular endothelial growth factor receptor 3 Ig (VEGFR-3-Ig) tumors (upper pair) and control LNM35 tumors (lower pair). (b) Lymph node (LN) volume and 95% confidence intervals (<span class="html-italic">p</span> = 0.070). (c,d) Histologic staining of lymph node sections from mice with the VEGFR−3−Ig−overexpressing and control tumors, respectively. Arrows = tumor cells in the lymph node. (<b>C</b>) The inhibition of the metastatic spread of orthotopic gastric AZL5G tumors by the systemic administration of anti−VEGFR−3 antibodies (AFL4) [<a href="#B143-targets-02-00015" class="html-bibr">143</a>]. (a) Control mouse (P; primary tumor, arrow; metastatic lymph node). (b): AFL4−treated mouse (P; primary tumor). (<b>D</b>) An assessment of lymphatic and blood vessel density in control and AFL4−treated mice [<a href="#B143-targets-02-00015" class="html-bibr">143</a>]. Immunohistochemistry of primary tumors for LYVE−1 (a,b) and CD31 (c,d) and schema of the vessel counts (e,f). Compared with the control group, the number of LYVE−1−positive lymphatic vessels (LVD) in the primary tumors in the AFL4−treated group is dramatically decreased (e, <span class="html-italic">p</span> < 0.05). In contrast, CD31−positive LYVE−1−negative microvessel density (MVD) was not significantly different between the control group and the AFL4−treated group (f, <span class="html-italic">p</span> = 0.84). In order to distinguish the figure and subfigure, the subfigures’ labels are replaced by lowercase letters in annotation.</p> Full article ">Figure 7
<p>The therapeutic potential of lymphatic system immunomodulatory capacity for lymphatic normalization. (<b>A</b>) VEGF−C/VEGFR-3 signaling increases the responsiveness of melanoma to immunotherapy [<a href="#B144-targets-02-00015" class="html-bibr">144</a>]. Tumor growth and survival of three different melanoma models treated with control (Iso) or aR3-blocking antibodies receiving different immunotherapies (arrows indicate times of administration). (a,b) B16-OVA/VC tumors treated with ATT in (a) WT (n ≥ 15) and (b) K14−VEGFR−3−Ig mice that lack dermal lymphatics (n = 4). (c–f) B16−OVA/VC tumors in WT mice treated with (c) ex vivo activated DCs (DC vax; n = 6), (d) 50 mg of CpG (n = 6), (e) 10 mg of OVA + 50 mg of CpG (n ≥ 8), and (f) 2 mg of Trp2 peptide−conjugated nanoparticles (NP-Trp2) + 50 mg of CpG (n = 7). (g) B16/VC tumors treated with NP-Trp2 + 50 mg of CpG (n = 6). (h) Tamoxifen-induced tumors in Braf<sup>V600E</sup>/Pten<sup>−/−</sup> mice treated with CpG + gp100 peptide (days 8 and 12) and anti−PD−1 antibody (day 12 and every 4 days thereafter). Each panel shows data from one (b–d,f,g), two (e), or three (a) independent experiments. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001 by two-tailed Student’s <span class="html-italic">t</span> test for growth curves and log-rank (Mantel-Cox) test for comparing survival curves. (<b>B</b>) The increased efficacy of immunotherapy in lymphangiogenic B16 melanomas depends on CCR7 signaling before therapy and the local activation and expansion of TILs after therapy [<a href="#B144-targets-02-00015" class="html-bibr">144</a>]. (a,b) B16-OVA/VC tumor–bearing mice treated with control IgG (Iso) or anti−VEGFR−3 (aR3)–blocking antibodies were euthanized 3 days after ATT, and tumor single-cell suspensions were analyzed by flow cytometry (n = 5). Quantification of overall naïve CD8<sup>+</sup> (CD45<sup>+</sup> CD8<sup>+</sup> CD44<sup>−</sup> CD62L<sup>+</sup>), effector CD8<sup>+</sup> (CD45<sup>+</sup> CD8<sup>+</sup> CD44<sup>+</sup> CD62L<sup>−</sup>), and OT-I (CD45<sup>+</sup> CD8<sup>+</sup> CD45.1<sup>+</sup>) T cells (a) in the tumor and (b) in the dLNs. (c) Tumor growth and survival curves of B16-OVA/VC tumor−bearing mice treated with anti-CCR7 (aCCR7), control IgG (Iso), or aR3 antibodies combined with ATT on day 9. CCR7 blockade was performed only before ATT (days 0, 3, and 6) (data pooled from two or more independent experiments, n ≥ 15 total). (d) Tumor growth curves of B16−OVA/VC tumor–bearing mice treated with control IgG (Iso) or aR3 antibodies received daily injections of the small molecular S1P inhibitor FTY720 starting on the same day as ATT was performed (day 9) (n ≥ 5). Statistics show differences between Iso + FTY720 and aR3 + FTY720 by one-way ANOVA. (e) Representative flow cytometry plots and (f) quantification of circulating CD4<sup>+</sup> and CD8<sup>+</sup> T cells (after B220 exclusion) in blood 26 days after tumor inoculation. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001 by two−tailed Student’s <span class="html-italic">t</span> test or one−way ANOVA and log-rank (Mantel−Cox) test for survival curves. (<b>C</b>) Dorsal MLVs are the main route for immune cell entry to draining CLNs [<a href="#B145-targets-02-00015" class="html-bibr">145</a>]. (a) Heat map of DEGs (Up, 219; Down, 100; power > 0.4). (b, c) Gene sets involved in lymphatic remodeling, fluid drainage, as well as inflammatory and immunological responses as shown by the representative upregulated pathways in GL261 tumor-associated and B16 tumor-associated MLECs compared to control MLECs (b), and heat map of DEGs enriched in the antigen processing and presentation pathway (c). (d) Left panels, treatment scheme and representative flow cytometry dot plots of DC trafficking from GL261 tumors to dCLNs in mice treated with Vehicle + Laser or Visudyne + Laser, determined by the quantity of CD11c<sup>+</sup>MHCII<sup>+</sup>FITC<sup>+</sup> cells in the dCLNs 24 h after intratumoral injection of FITC-labeled latex beads. Right panel, quantification of Bead<sup>+</sup> DCs in the dCLNs of mice treated with Vehicle + Laser or Visudyne + Laser. (e) Immunoprecipitation of secreted VEGF−C protein (arrow) in conditioned medium from GL261-Vector, GL261-VEGF-C, B16-Vector, and B16−VEGF−C cells. (f) Left panels, LYVE-1 and CCL21 staining of MLVs in mice bearing Empty and VEGF-C-overexpressing GL261 tumors in the striatum (scale bars, 100 µm in wide-fields; 50 µm in insets). Right panels, quantification of the percentage area of LYVE-1 and CCL21 (n = 10). (g) Left panels, treatment scheme and representative flow cytometry dot plots of DC trafficking in the dCLNs of mice bearing GL261 tumors overexpressing Vector or VEGF−C. Right panel, quantification of bead<sup>+</sup> DCs in dCLNs (n = 10). (h) Left panels, treatment scheme and representative flow cytometry dot plots of DC trafficking in the dCLNs of GL261 tumor-bearing mice treated with CCL21 (αCCL21)- or IgG (Iso)-blocking antibodies. Right panel, quantification of bead<sup>+</sup> DCs in dCLNs (n = 10). * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. (<b>D</b>) A high level of tumor-derived VEGF-C improves anti-PD-1/CTLA-4 efficacy [<a href="#B145-targets-02-00015" class="html-bibr">145</a>]. (a) Survival of mice with striatal Vector- or VEGF-Coverexpressing GL2161 tumors following the administration of anti-PD-1/CTLA-4 or IgG controls (n = 15). (b) Representative T2-weighted single brain slices from mice with intracranial injection of GL261 cells overexpressing Vector or VEGF-C (n = 6). (c) Tumor volumes in mice with striatal injection of GL261 cells overexpressing Vector or VEGF-C (n = 6). (d,e) Quantification of CD8<sup>+</sup>Ki67<sup>+</sup> T cells (d) and CD4<sup>+</sup>Foxp3<sup>+</sup> T cells (e) as percentages of overall CD45<sup>+</sup> cells in tumors and in dCLNs on day 14 after inoculation (n = 12 in each). (f) Ratios of CD8<sup>+</sup>Ki67<sup>+</sup> T cells to Tregs in tumors and in dCLNs. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. In order to distinguish the figure and subfigure, the subfigures’ labels are replaced by lowercase letters in annotation.</p> Full article ">Figure 8
<p>Normalization of the matrix. (<b>A</b>) The effect of collagenase on oncolytic viral therapy [<a href="#B147-targets-02-00015" class="html-bibr">147</a>]. (<b>B</b>) The effect of collagenase on MGH2-induced tumor growth delay [<a href="#B147-targets-02-00015" class="html-bibr">147</a>]. (<b>C</b>) The buckling of a collagen fiber in a relaxin-treated tumor [<a href="#B149-targets-02-00015" class="html-bibr">149</a>]. (<b>D</b>) A quantitative analysis of collagen fiber length and the area of colocalization between stromal cells and fibers [<a href="#B149-targets-02-00015" class="html-bibr">149</a>]. (a,b) The end-to-end fiber length (a) and the area of overlap between stromal cells and collagen fibers (b) was determined over 4 d in control groups as well as those treated with relaxin, relaxin and b1 integrin antibody (R + b1) and relaxin and GM6001 (R + GM). Shown are the averaged differences between day 1 and days 2 (D2-1), 3 (D3-1) and 4 (D4-1). * <span class="html-italic">p</span> < 0.05. (<b>E</b>) Blocking TGF-β signaling improves the intratumoral distribution of doxorubicin in orthotopic mammary carcinoma models [<a href="#B153-targets-02-00015" class="html-bibr">153</a>]. (a,b) Representative images of doxorubicin intratumoral distribution in 4T1 (a) and 4T1–sTβRII (b) tumors. Green, FITC–lectin-labeled perfused vessels; red, fluorescent doxorubicin; blue, DAPI. (c) Quantification of the fraction of tumor area positive for doxorubicin (n = 12 sections, with 3 sections per tumor). * <span class="html-italic">p</span> < 0.001. (<b>F</b>) Blocking TGF-β signaling decreases collagen I content and improves Doxil tissue penetration [<a href="#B153-targets-02-00015" class="html-bibr">153</a>]. (a) Representative images and quantification of collagen I immunofluorescent staining in 4T1 and 4T1–sTβRII tumors. Red, collagen I staining; blue, DAPI (×20). (b) Representative images and quantification of Doxil intratumoral distribution in 4T1 and 4T1–sTβRII tumors. Green, FITC-lectin labeled perfused vessels; red, fluorescent doxorubicin; blue, DAPI (n = 12 sections, with 3 sections per tumor). * <span class="html-italic">p</span> < 0.001. (<b>G</b>) Blocking TGF-β signaling enhances Doxil efficacy in orthotopic mammary carcinoma models [<a href="#B153-targets-02-00015" class="html-bibr">153</a>]. (a and b) Primary tumor growth of 4T1 and 4T1–sTβRII tumors, with or without Doxil treatment (a) and 4T1 (b) and MDA-MB-231 (c) tumors treated with saline (control), Doxil alone (9 mg/kg, weekly), 1D11 alone (5 mg/kg, three times a week), or combined Doxil and 1D11 (n = 8 in all groups). In order to distinguish the figure and subfigure, the subfigures’ labels are replaced by lowercase letters in annotation.</p> Full article ">Figure 9
<p>Biochemical therapies for breaking the safe haven. (<b>A</b>) The brain penetration of ABT-888 is limited by Abcb1 and Abcg2 [<a href="#B162-targets-02-00015" class="html-bibr">162</a>]. (a) ABT-888 plasma concentrations, brain concentrations, and brain-to-plasma ratios following intravenous administration of 10 mg/kg of ABT-888 (n = 5/time point/strain). (b) ABT-888 levels following 10 mg/kg p.o. administered to wild-type and Abcb1a/1b<sup>−/−</sup>; Abcg2<sup>−/−</sup> mice with/without co-administration of 100 mg/kg elacridar p.o. Blood samples were collected from the tail at 15 min, 1, 2, and 4 h (n = 8/strain). Brain samples were harvested at 4 h after drug administration. **** <span class="html-italic">p</span> < 0.0001 compared with wild-type. (<b>B</b>) The inhibition of Abcb1 and Abcg2 improve efficacy of ABT-888 + TMZ treatment [<a href="#B162-targets-02-00015" class="html-bibr">162</a>]. (a) Efficacy of TMZ versus TMZþABT-888 treatment against intracranial p53; p16<sup>Ink4a</sup>/p19<sup>Arf</sup>; K-Ras<sup>v12</sup>; LucR GBM652457 cells injected into wild-type mice. (b) Same setup but now injected into Abcb1a/1b<sup>−/−</sup>; Abcg2<sup>−/−</sup>(KO) mice. (c) Efficacy of TMZ + ABT-888 with and without elacridar in both WT and KO mice. (d) Efficacy of TMZ or TMZ + ABT-888 with or without elacridar against (lentivirally induced) spontaneous p53; p16<sup>Ink4a</sup>/p19<sup>Arf</sup>; K-Ras<sup>v12</sup>; LucR tumors. TMZ p.o. at 100 mg/kg every day alone or concurrently with ABT-888 p.o. at 10 mg/kg twice a day and/or elacridar p.o. at 100 mg/kg every day 15 min before TMZ for 5 days. Left, relative tumor growth curves. Right, Kaplan–Meier analysis of survival. (<b>C</b>) PTEN-deficient tumors are more sensitive to ABT-888 + TMZ treatment [<a href="#B162-targets-02-00015" class="html-bibr">162</a>]. (a) Kaplan–Meier analysis of PTEN deletion (≤1.8 copies) on overall survival of patients with glioblastoma. (b) Sensitivity (in vitro) of two panels of glioblastoma cell lines of different genetic backgrounds exposed to 100 μmol/L TMZ and increasing concentrations of ABT-888 for 5 days. (c) Efficacy of ABT-888 in combination with TMZ against Pten;p16<sup>Ink4a</sup>/p19<sup>Arf</sup>;K-Ras<sup>v12</sup>;LucR GBM696677 cells injected intracranially into WT or Abcb1a/1b<sup>−/−</sup>;Abcg2<sup>−/−</sup> (KO) mice and (d) spontaneous Pten;p16<sup>Ink4a</sup>/p19<sup>Arf</sup>; K-Ras<sup>v12</sup>;LucR tumors. TMZ (100 mg/kg p.o. every day) alone or concurrently with ABT-888 (10 mg/kg p.o. twice a day) for 5 days. C and d (right), Kaplan-Meier analysis of survival. (<b>D</b>) The brain concentration of paclitaxel (10 mg/kg) in mice at 4 h after administration of paclitaxel alone and in combination with different (putative) inhibitors of Pgp (Pgp knockout mice were used as a reference for “complete” inhibition of Pgp) [<a href="#B163-targets-02-00015" class="html-bibr">163</a>]. In order to distinguish the figure and subfigure, the subfigures’ labels are replaced by lowercase letters in annotation.</p> Full article ">Figure 10
<p>Physical therapies for breaking the safe haven. (<b>A</b>) FUS enhances doxorubicin (Dox) penetration and promotes convective transport in BT474-Gluc brain tumors [<a href="#B164-targets-02-00015" class="html-bibr">164</a>]. (a) Sequential intravital multiphoton microscopy of Dox autofluorescence. (b) Temporal evaluation of Dox extravasation with and without FUS-BTB disruption. Cv and Ce are the mean pixel intensity of the vessel and the extravascular space, respectively. The maximum mean fluorescence for the FUS and non-FUS was 0.52 ± 0.15 and 0.07 ± 0.02, a sevenfold difference. (c) Dox penetration from a line profile perpendicular to vessel wall (red dotted arrow in a). The plot shows the normalized maximum intensity projection (MIP) across the series of images. The dotted line shows a regression fitted to the data from four different animals for each condition (non-FUS and FUS). (d) Representative data of the temporal evolution of the normalized intensity of the line profile (red dotted arrow in a). For consistency in the notation of the experiments/modeling, Cv is the Dox intensity/concentration in the vessel, Ce is the Dox intensity/concentration in the extracellular/interstitial space. (<b>B</b>) FUS-BTB disruption increases early extravasation and penetration of T-DM1 in BT474-Gluc brain tumors [<a href="#B164-targets-02-00015" class="html-bibr">164</a>]. (a) Representative microscopy data of TDM1 extravasation with and without FUS at 4 h and 5 d. (b) Quantification of the T-DM1 extravasation (Left) and penetration (Right) with and without FUS at 4 h (Upper) and 5 d (Lower) posttreatment. Parametric Student’s <span class="html-italic">t</span> test for <span class="html-italic">p</span> < 0.05. (<b>C</b>) The quantification of transvascular transport via mathematical modeling demonstrates a multifold increase in the effective diffusion coefficient (4.3-fold) and in hydraulic conductivity (4.5–fold) after FUS-BTB disruption [<a href="#B164-targets-02-00015" class="html-bibr">164</a>]. (a) Schematic illustrating the transport of the anticancer agents from the vessel to the interstitial space along with the studied model parameters and agent-specific cellular uptake model equations. (Upper) Convection–diffusion–reaction model following Michaelis–Menten kinetics with binding of doxorubicin to DNA (Vb). (Lower) Convection–diffusion–reaction model for T-DM1. Excellent fit was observed for both doxorubicin and T-DM1. (b, Upper) The time dependence of doxorubicin extravasation from the fitted and experimental data for non–FUS and FUS–BBB/BTB disruption groups. (b, Lower) Parameter fit methodology for T–DM1 and fitted data from two different experiments. The fitted vascular and interstitial effective porosity (fraction of surface area occupied by pores) from the doxorubicin model was used as input to the T-DM1 fitting (i.e., same animal model). (c) Normalized parameter fit for non-FUS and FUS-BBB/BTB disruption groups (Upper, doxorubicin; Lower, T-DM1). The values for each parameter were normalized to maximum to be displayed on the same plot. The exact numbers and their units are shown in <a href="#targets-02-00015-t001" class="html-table">Table 1</a> and <a href="#targets-02-00015-t002" class="html-table">Table 2</a> for doxorubicin and T-DM1, respectively. (<b>D</b>) The diminished interaction of intracellular scaffolding proteins ZO-1 and occludin as a result of ultrasound treatment [<a href="#B166-targets-02-00015" class="html-bibr">166</a>]. (a) Western Blot analysis on whole brain tissue lysates (WTL) shows that ZO-1 and occludin protein levels are not changed in response to ultrasound treatment. (b), Co-immunoprecipitation of occludin and ZO-1. The amount of occludin co-precipitating with ZO-1 in the presence of US-treatment is reduced when compared to non-sonicated brains (-US). (<b>E</b>) Ultrasound in the presence of microbubbles increases the activity of the Akt signaling pathway, while the activity of MAPK signaling remains unchanged [<a href="#B166-targets-02-00015" class="html-bibr">166</a>]. Brains were removed 1.5 h (a) or 24 h (b) post-sonication and snap-frozen using liquid nitrogen. Brain tissue regions with trypan blue leakage in the sonicated hemisphere (+US) and the equivalent area from opposite hemisphere (-US) were then homogenized with RIPA lysis buffer. Equal amounts of extracted proteins were analyzed by western blotting for the indicated proteins. (c) Graphical representation of three independent experiments illustrating the marked increase in pAkt (Ser473), pAkt (Thre308) and pGSK3b (Ser9) 1.5 hrs after sonication treatment. The star (*) represents <span class="html-italic">p</span> < 0.05 for -US versus +US. (<b>F</b>) Increased phosphorylation of Akt and GSK3β in neuronal cells of sonicated rat brain regions [<a href="#B166-targets-02-00015" class="html-bibr">166</a>]. (a) Panel [i] shows a control section without application of any primary antibodies. Panel [ii] represents a direct staining of the astrocytes with Alexa-fluor488-conjugated GFAP (green, right arrow). Panels [iii] and [iv] represent immunofluorescence staining with pAkt (red, curved arrow) and GFAP (green, right arrow) in non-sonicated (-US) as well as sonicated (+US) hemispheres respectively. Panels [v] and [vi] illustrate co-staining of pGSK3b (red, pentagon) and GFAP (green, right arrow) in ‘-US’ and ‘+US’ brain regions respectively. (b) Panels [ii] and [iii] represent neuronal cells morphology as directly stained with Alexa-Fluor 488-conjugated NeuN (green, lightning bolt). Co-staining of pAkt (red, curved arrow) with NeuN (green) and also pGSK3b (red, pentagon) with NeuN (green) are shown in panels [iii, iv] and [v, vi] respectively. The regions of the brain sections with IgG extravasation (arrow head) and their surrounding neuronal cells with elevated levels of pAkt and pGSK3b are shown in panels [iv, vi]. (<b>G</b>) Representative Evans Blue (EB) dye staining in animal brains after FUS-BBB opening [<a href="#B167-targets-02-00015" class="html-bibr">167</a>]. (<b>H</b>) (a) The TMZ concentration (mean ± STD) at 2 h after TMZ administration obtained from plasma and brain tissues from each experimental group. (b) The estimated time (in hours) for TMZ to degrade to 50% of the peak level [<a href="#B167-targets-02-00015" class="html-bibr">167</a>]. (<b>I</b>) (a) The average fluorescence intensity and (b) average area of fluorescence for the 3, 70, 500, and 2000 kDa dextrans sonicated at 0.31, 0.51, and 0.84 MPa. The pressure threshold for significant increases in both fluorescence and the area of fluorescence was 0.51 MPa for 3 and 70 kDa dextrans. However, it increased to 0.84 MPa for the 500 and 2000 kDa dextrans [<a href="#B169-targets-02-00015" class="html-bibr">169</a>]. (<b>J</b>) Microscopic examination of (a,c) left (sonicated) and (b,d) the corresponding right (nonsonicated) hippocampi in hemotoxylin and eosin-stained, 6 μm-thick horizontal sections [<a href="#B169-targets-02-00015" class="html-bibr">169</a>].</p> Full article ">