The radiopharmaceutical radium-223 has immunomodulatory effects in patients and facilitates anti-programmed death receptor-1 therapy in murine models of bone metastatic prostate cancer.

BACKGROUND & PURPOSE: Radium-223 (Ra223) improves survival in metastatic prostate cancer (mPC), but its impact on systemic immunity is unclear, and biomarkers of response are lacking. We examined markers of immunomodulatory activity during standard clinical Ra223 and studied the impact of Ra223 on response to immune checkpoint inhibition (ICI) in preclinical models. MATERIALS & METHODS: We conducted a single-arm biomarker study of Ra223 in 22 bone mPC patients. We measured circulating immune cell subsets and a panel of cytokines before and during Ra223 therapy and correlated them with overall survival (OS). Using two murine mPC models-orthotopic PtenSmad4-null and TRAMP-C1 grafts in syngeneic immunocompetent mice-we tested the efficacy of combining Ra223 with ICI. RESULTS: Above-median level of IL-6 at baseline was associated with a median OS of 358 versus 947 days for below levels; p = 0.044, from the log-rank test. Baseline PlGF and PSA inversely correlated with OS (p = 0.018 and p = 0.037, respectively, from the Cox model). Ra223 treatment was associated with a mild decrease in some peripheral immune cell populations and a shift in the proportion of MDSCs from granulocytic to myeloid. In mice, Ra223 increased the proliferation of CD8+ and CD4+ helper T cells without leading to CD8+ T cell exhaustion in the mPC lesions. In one of the models, combining Ra223 and anti-PD-1 antibody significantly prolonged survival, which correlated with increased CD8+ T cell infiltration in tumor tissue. CONCLUSION: The inflammatory cytokine IL-6 and the angiogenic biomarker PlGF at baseline were promising outcome biomarkers after standard Ra223 treatment. In mouse models, Ra223 increased intratumoral CD8+ T cell infiltration and proliferation and could improve OS when combined with anti-PD-1 ICI.

LC-MS/MS determination of GTS-201, a dipeptide mimetic of the brain-derived neurotrophic factor, and neurotransmitter metabolites with application to a pharmacokinetic study in rats.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family with diverse psychopharmacological effects including antidepressant and anxiolytic actions. However, the clinical use of BDNF is limited due to its poor pharmacokinetic properties. The development of low-molecular-weight BDNF mimetics passing through the blood-brain barrier is an emerging strategy for improved managing psychiatric diseases. The present study characterizes a novel dipeptide mimetic of the 2nd BDNF loop named GTS-201, which exhibits psychotropic properties in experimental animal models of anxiety and alcohol dependence. The aim of this work was to study the pharmacokinetics of GTS-201 in rats at a saturating dosage of 5 mg/kg applied by the intraperitoneal route and to characterize the effects on neurotransmitter levels in the blood and brain. The maximum concentration (Cmax) of GTS-201 in the plasma (867 ± 69 ng/ml) was recorded at 35 ± 7.7 min after administration (Tmax) with a half-elimination period (T1/2) of 19.5 ± 1.8 min, while in the brain tissue Cmax was 14.92 ± 3.11 ng/ml, Tmax was 40.0 ± 7.7 min and T1/2 were 87.5 ± 12.7 min. The relative tissue availability of the GTS-201 for the brain reached 2.9%. At the dose applied, GTS-201 induced a significant increase of serotonin (5-fold) and dopamine levels in the brain tissue (8-fold) along with a decrease in cortisol content in blood plasma 45 min after acute administration. In summary, GTS-201 crosses the blood-brain barrier after acute administration and affects the activity of serotonergic and dopaminergic systems, which may underlie its neuropsychotropic effects described previously.

Inhibition of CXCR4 Enhances the Efficacy of Radiotherapy in Metastatic Prostate Cancer Models.

Radiotherapy (RT) is a standard treatment for patients with advanced prostate cancer (PCa). Previous preclinical studies showed that SDF1α/CXCR4 axis could mediate PCa metastasis (most often to the bones) and cancer resistance to RT. We found high levels of expression for both SDF1α and its receptor CXCR4 in primary and metastatic PCa tissue samples. In vitro analyses using PCa cells revealed an important role of CXCR4 in cell invasion but not radiotolerance. Pharmacologic inhibition of CXCR4 using AMD3100 showed no efficacy in orthotopic primary and bone metastatic PCa models. However, when combined with RT, AMD3100 potentiated the effect of local single-dose RT (12 Gy) in both models. Moreover, CXCR4 inhibition also reduced lymph node metastasis from primary PCa. Notably, CXCR4 inhibition promoted the normalization of bone metastatic PCa vasculature and reduced tissue hypoxia. In conclusion, the SDF1α/CXCR4 axis is a potential therapeutic target in metastatic PCa patients treated with RT.

Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases.

The recent landmark Phase III clinical trial with a VEGF-specific antibody suggests that antiangiogenic therapy must be combined with cytotoxic therapy for the treatment of solid tumors. However, there are no guidelines for optimal scheduling of these therapies. Here we show that VEGFR2 blockade creates a "normalization window"--a period during which combined radiation therapy gives the best outcome. This window is characterized by an increase in tumor oxygenation, which is known to enhance radiation response. During the normalization window, but not before or after it, VEGFR2 blockade increases pericyte coverage of brain tumor vessels via upregulation of Ang1 and degrades their pathologically thick basement membrane via MMP activation.

Vascular damage in tumors: a key player in stereotactic radiation therapy?

The use of stereotactic radiation therapy (SRT) for cancer treatment has grown in recent years, showing excellent results for some tumors. The greatly increased doses per fraction in SRT compared to conventional radiotherapy suggest a 'new biology' that determines treatment outcome. Proposed mechanisms include significant damage to tumor blood vessels and enhanced antitumor immune responses, which are also vasculature-dependent. These ideas are mostly based on the results of radiation studies in animal models because direct observations in humans are limited. However, even preclinical findings are somewhat incomplete and result in ambiguous conclusions. Current evidence of vasculature-related mechanisms of SRT is reviewed. Understanding them could result in better optimization of SRT alone or in combination with immune or other cancer therapies.

Insights into the Cardiotoxic Effects of Veratrum Lobelianum Alkaloids: Pilot Study.

Jervine, protoveratrine A (proA), and protoveratrine B (proB) are Veratrum alkaloids that are presented in some remedies obtained from Veratrum lobelianum, such as Veratrum aqua. This paper reports on a single-center pilot cardiotoxic mechanism study of jervine, proA, and proB in case series. The molecular aspects were studied via molecular dynamic simulation, molecular docking with cardiac sodium channel NaV1.5, and machine learning-based structure-activity relationship modeling. HPLC-MS/MS method in combination with clinical events were used to analyze Veratrum alkaloid cardiotoxicity in patients. Jervine demonstrates the highest docking score (-10.8 kcal/mol), logP value (4.188), and pKa value (9.64) compared with proA and proB. Also, this compound is characterized by the lowest calculated IC50. In general, all three analyzed alkaloids show the affinity to NaV1.5 that highly likely results in cardiotoxic action. The clinical data of seven cases of intoxication by Veratrum aqua confirms the results of molecular modeling. Patients exhibited nausea, muscle weakness, bradycardia, and arterial hypotension. The association between alkaloid concentrations in blood and urine and severity of patient condition is described. These experiments, while primary, confirmed that jervine, proA, and proB contribute to cardiotoxicity by NaV1.5 inhibition.

Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer.

The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors.

The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis.

Gliomas are the most common primary tumours of the central nervous system, with nearly 15,000 diagnosed annually in the United States and a lethality approaching 80% within the first year of glioblastoma diagnosis. The marked induction of angiogenesis in glioblastomas suggests that it is a necessary part of malignant progression; however, the precise molecular mechanisms underlying the regulation of brain tumour growth and angiogenesis remain unresolved. Here we report that a candidate tumour suppressor gene, ING4, is involved in regulating brain tumour growth and angiogenesis. Expression of ING4 is significantly reduced in gliomas as compared with normal human brain tissue, and the extent of reduction correlates with the progression from lower to higher grades of tumours. In mice, xenografts of human glioblastoma U87MG, which has decreased expression of ING4, grow significantly faster and have higher vascular volume fractions than control tumours. We show that ING4 physically interacts with p65 (RelA) subunit of nuclear factor NF-kappaB, and that ING4 regulates brain tumour angiogenesis through transcriptional repression of NF-kappaB-responsive genes. These results indicate that ING4 has an important role in brain tumour pathogenesis.

Human tumor xenografts recurring after radiotherapy are more sensitive to anti-vascular endothelial growth factor receptor-2 treatment than treatment-naive tumors.

The effects of antiangiogenic therapy on tumors relapsing after irradiation are not known. To this end, we irradiated human tumors growing s.c. in nude mice with a single dose of 20 or 30 Gy. Compared with primary (treatment-naive) xenografts, the growth rate of recurrent tumors was 1.6-fold slower, which is consistent with the known "tumor bed effect." For similar size tumors, recurrences had fewer functional vessels, a reduced vessel coverage by perivascular cells, and were more necrotic. Placenta growth factor concentration was significantly lower in relapses, whereas vascular endothelial growth factor (VEGF) levels were similar between primary and recurrent tumors. On the other hand, fibrillar collagen deposition was significantly increased in recurrent tumors. This radiation-induced fibrosis was partially responsible for the slower growth of recurrences; the i.t. injection of collagenase increased the growth rate of tumor relapses without affecting primary tumor growth. The mouse-specific VEGF receptor 2-blocking antibody DC101 induced a 2.2-fold longer growth delay in recurrent tumors compared with treatment-naive tumors. DC101 significantly decreased the interstitial fluid pressure and did not change the functional vessel density and perivascular cell coverage in both tumor variants. Interestingly, DC101 induced a rapid (2 days after treatment initiation) and significant decrease in tumor cell proliferation in recurrent but not in primary tumors. Thus, our results show that the stromal compartment and the response to antiangionenic therapy of primary and in-field recurrent tumors are significantly different. Our findings suggest that antiangiogenic agents could be effective in the treatment of patients with relapses after radiotherapy.

Inter- and intramouse heterogeneity of radiation response for a growing paired organ.

An intensive search for predictive markers of individual radiation response of apparently normal tissues in cancer patients is in progress at the genetic and epigenetic levels. However, the relative impact of variability at these levels is not clear. Experimental results obtained in inbred rodents, which have significantly reduced genetic heterogeneity relative to a population of human patients, may help to clarify this issue. We investigated a paired-organ mouse system in a strain of inbred mice to evaluate the intermouse variability of normal tissue radiation response, singled out from measurement errors and stochastic effects. The legs of 5-day-old C3H mice were homogeneously gamma-irradiated with a range of single doses. The lengths of the right and left tibiae were measured in 30 kVp X-ray images taken at the time of irradiation and at 84 days postirradiation. The dose-effect curves were smooth and well defined, with bone growth retardation evident at approximately 14 Gy and higher, and were marginally gender-dependent. The intramouse (left compared to right) variability of the tibia length on day 89, which characterized stochastic effects, was not distinguishable from the measurement error for doses less than 16-18 Gy and slightly exceeded measurement errors only at the largest doses of 20-22 Gy. The corresponding intermouse variability was greater than the measurement error and stochastic effects at all doses used. Interestingly, the total variability, judged by the gamma(50) values of approximately 7 we obtained, was similar to that reported for severe late reactions in human normal tissue. If the variations of response determined by epigenetic events in human patients free of known factors associated with altered radiation sensitivity are comparable to those observed in this mouse model, our results imply a relatively low power of genetic approaches alone to predict individual side effects in radiotherapy.

Validated HPLC-MS/MS method for quantification of ethylmethylhydroxypyridine succinate in rat brain and its application to a pharmacokinetic study.

2-ethyl-6-methyl-3-hydroxypyridine (EMHP) succinate is the original antioxidant and antihypoxic drug commonly prescribed in Russia. The objective of this study was to develop a rapid, simple and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for EMHP quantitation in rat brain tissue with the use of a bead beating homogenizer. The comparison between two approaches to brain tissue preparation was performed, when spiking the blank brain tissue with EMHP reference standard and internal standard (IS) before and after homogenization step. Chromatographic separation was achieved using Zorbax Eclipse Plus C18 column (1.8 µm, 2.1 × 50 mm) and elution was performed with the mobile phase, consisting of 10 mM of ammonium formate aqueous solution with 0.1% formic acid as solvent A and 0.1% formic acid in methanol as solvent B [44%(А):56%(В), v/v]. Flow rate was 0.4 mL/min and the total run time for each sample analysis was 2.0 min. EMHP and amantadine, IS of this study, were analyzed in positive ionization mode. Ion transitions of m/z 138.0 → 123.0 for EMHP and m/z 152.0 → 135.0 for amantadine were selected in multiple reaction monitoring mode. The developed method for EMHP determination in rat brain samples was validated for selectivity, linearity, accuracy, precision, matrix effects, and stability. The lower and upper limits of quantification were determined to be 1 and 1500 ng/g, respectively. The developed and validated HPLC-MS/MS method was successfully applied to determine EMHP concentrations in rat brain tissue following the intraperitoneal administration at a dose of 3.4 mg/kg.

Secretory leukocyte protease inhibitor (SLPI) as a potential target for inhibiting metastasis of triple-negative breast cancers.

SLPI has been implicated in the progression and metastasis of certain cancers. However, the effects of SLPI seem to be tumor-specific and the mechanisms remain poorly defined. Here, we demonstrate that highly metastatic, triple-negative breast cancer (TNBC) 4T1 cells secreted more SLPI compared to their non-metastatic counterparts. Furthermore, SLPI secretion directly correlated with spontaneous lung metastasis from 4T1 tumors orthotopically implanted in mice. Consistent with our experimental results, we also found that higher SLPI expression levels correlate with worse clinical outcome in basal/TNBC patients. Using high-throughput screening we identified a novel compound, C74, which significantly inhibits SLPI secretion. C74 administration in our mouse model slows the growth of primary 4T1 tumors and inhibits their dissemination to the lung. We also discovered that SLPI physically interacts with the retinoblastoma tumor suppressor protein (Rb) and releases FoxM1 from the Rb-FoxM1 complex, which may activate FoxM1 target genes involved in breast cancer metastasis.

Combined vascular endothelial growth factor-targeted therapy and radiotherapy for rectal cancer: theory and clinical practice.

Despite the routine use of adjuvant and neoadjuvant chemoradiotherapy, patients with advanced rectal tumors experience significant rates of treatment failure and disease recurrence. Resistance to radiation is a particular problem. Adding a vascular endothelial growth factor (VEGF)-targeted therapy may improve outcomes in these patients. Epidemiologic studies have shown that tumor expression of VEGF predicts disease recurrence and lower overall survival in patients treated with radiation. In tumor xenograft models in mice, VEGF-targeted agents increase the response to radiation, with a greater probability of tumor control and a greater delay in tumor growth. In addition to killing cancer cells indirectly by damaging tumor blood vessels (antivascular effect), VEGF-targeted therapy may sensitize tumors to radiation through two mechanisms: by normalizing the tumor vasculature, leading to greater tumor oxygenation, and thereby increasing the cytotoxicity of radiation to cancer cells, and by increasing the radiosensitivity of tumor-associated endothelial cells. In addition, anti-VEGF agents may inhibit the regrowth of tumors after radiation by decreasing the number of circulating endothelial cells and endothelial progenitor cells. A phase I dose-escalation study has shown the safety of bevacizumab at a dose of 5 mg/kg in combination with 5-fluorouracil and radiation in patients with rectal carcinoma, and has provided evidence of both vascular normalization and antivascular mechanisms. Phase II evaluation of bevacizumab in this setting is under way.

Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors.

Elevated interstitial fluid pressure, a hallmark of solid tumors, can compromise the delivery of therapeutics to tumors. Here we show that blocking vascular endothelial growth factor (VEGF) signaling by DC101 (a VEGF-receptor-2 antibody) decreases interstitial fluid pressure, not by restoring lymphatic function, but by producing a morphologically and functionally "normalized" vascular network. We demonstrate that the normalization process prunes immature vessels and improves the integrity and function of the remaining vasculature by enhancing the perivascular cell and basement membrane coverage. We also show that DC101 induces a hydrostatic pressure gradient across the vascular wall, which leads to a deeper penetration of molecules into tumors. Thus, vascular normalization may contribute to the improved survival rates in tumor-bearing animals and in colorectal carcinoma patients treated with an anti-VEGF antibody in combination with cytotoxic therapies.

Blockade of MMP14 activity in murine breast carcinomas: implications for macrophages, vessels, and radiotherapy.

BACKGROUND: Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects. METHODS: Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor ß (TGFß) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided. RESULTS: DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFß and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors. CONCLUSION: MMP14 blockade decreased immunosuppressive TGFß, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors.

Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels.

Cancer and stromal cells actively exert physical forces (solid stress) to compress tumour blood vessels, thus reducing vascular perfusion. Tumour interstitial matrix also contributes to solid stress, with hyaluronan implicated as the primary matrix molecule responsible for vessel compression because of its swelling behaviour. Here we show, unexpectedly, that hyaluronan compresses vessels only in collagen-rich tumours, suggesting that collagen and hyaluronan together are critical targets for decompressing tumour vessels. We demonstrate that the angiotensin inhibitor losartan reduces stromal collagen and hyaluronan production, associated with decreased expression of profibrotic signals TGF-ß1, CCN2 and ET-1, downstream of angiotensin-II-receptor-1 inhibition. Consequently, losartan reduces solid stress in tumours resulting in increased vascular perfusion. Through this physical mechanism, losartan improves drug and oxygen delivery to tumours, thereby potentiating chemotherapy and reducing hypoxia in breast and pancreatic cancer models. Thus, angiotensin inhibitors -inexpensive drugs with decades of safe use - could be rapidly repurposed as cancer therapeutics.

Neovascularization after irradiation: what is the source of newly formed vessels in recurring tumors?

Local relapse of tumors after radiation therapy remains a challenge in oncology. To devise rational approaches for preventing this relapse, we have to improve our understanding of how new vessels form in previously irradiated tumors. We propose that tumor regrowth after local irradiation is dependent on blood vessel formation by local endothelial cells without the need for recruitment of endothelial precursor cells from distant nonirradiated tissues or bone marrow. We also suggest that infiltrating myeloid bone marrow-derived cells promote survival of local endothelial cells during the early period after irradiation and angiogenesis during the later stage of tumor regrowth, both via paracrine mechanisms.

CXCL12 (SDF1alpha)-CXCR4/CXCR7 pathway inhibition: an emerging sensitizer for anticancer therapies?

Addition of multiple molecularly targeted agents to the existing armamentarium of chemotherapeutics and radiotherapies represents a significant advance in the management of several advanced cancers. In certain tumor types with no efficacious therapy options, these agents have become the first line of therapy, for example, sorafenib in advanced hepatocellular carcinoma or bevacizumab in recurrent glioblastoma. Unfortunately, in many cases, the survival benefits are modest, lasting only weeks to a few months. Moreover, they may not show benefit in patients with localized disease (i.e., in the adjuvant setting). Recent studies have provided increasing evidence that activation of the chemokine CXCL12 (SDF1α) pathway is a potential mechanism of tumor resistance to both conventional therapies and biological agents via multiple complementary actions: (i) by directly promoting cancer cell survival, invasion, and the cancer stem and/or tumor-initiating cell phenotype; (ii) by recruiting "distal stroma" (i.e., myeloid bone marrow-derived cells) to indirectly facilitate tumor recurrence and metastasis; and (iii) by promoting angiogenesis directly or in a paracrine manner. Here, we discuss recent preclinical and clinical data that support the potential use of anti-CXCL12 agents (e.g., AMD3100, NOX-A12, or CCX2066) as sensitizers to currently available therapies by targeting the CXCL12/CXCR4 and CXCL12/CXCR7 pathways.

Recruitment of myeloid but not endothelial precursor cells facilitates tumor regrowth after local irradiation.

Tumor neovascularization and growth might be promoted by the recruitment of bone marrow-derived cells (BMDC), which include endothelial precursor cells and "vascular modulatory" myelomonocytic (CD11b+) cells. BMDCs may also drive tumor regrowth after certain chemotherapeutic and vascular disruption treatments. In this study, we evaluated the role of BMDC recruitment in breast and lung carcinoma xenograft models after local irradiation (LI). We depleted the bone marrow by including whole-body irradiation (WBI) of 6 Gy as part of a total tumor dose of 21 Gy, and compared the growth delay with the one achieved after LI of 21 Gy. In both models, the inclusion of WBI induced longer tumor growth delays. Moreover, WBI increased lung tumor control probability by LI. Exogenous delivery of BMDCs from radiation-naïve donors partially abrogated the WBI effect. Myeloid BMDCs, primarily macrophages, rapidly accumulated in tumors after LI. Intratumoral expression of stromal-derived factor 1alpha (SDF-1alpha), a chemokine that promotes tissue retention of BMDCs, was noted 2 days after LI. Conversely, treatment with an inhibitor of SDF-1alpha receptor CXCR4 (AMD3100) with LI significantly delayed tumor regrowth. However, when administered starting from 5 days post-LI, AMD3100 treatment was ineffective. Lastly, with restorative bone marrow transplantation of Tie2-GFP-labeled BMDC population, we observed an increased number of monocytes but not endothelial precursor cells in tumors that recurred following LI. Our results suggest that an increase in intratumoral SDF-1alpha triggered by LI recruits myelomonocytes/macrophages which promotes tumor regrowth.