Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients.

BACKGROUND: Tregs trafficking is controlled by CXCR4. In Renal Cell Carcinoma (RCC), the effect of the new CXCR4 antagonist, R54, was explored in peripheral blood (PB)-Tregs isolated from primary RCC patients. METHODS: PB-Tregs were isolated from 77 RCC patients and 38 healthy donors (HDs). CFSE-T effector-Tregs suppression assay, IL-35, IFN-γ, IL-10, TGF-ß1 secretion, and Nrp-1+Tregs frequency were evaluated. Tregs were characterised for CTLA-4, PD-1, CD40L, PTEN, CD25, TGF-ß1, FOXP3, DNMT1 transcriptional profile. PTEN-pAKT signalling was evaluated in the presence of R54 and/or triciribine (TCB), an AKT inhibitor. Methylation of TSDR (Treg-Specific-Demethylated-Region) was conducted. RESULTS: R54 impaired PB-RCC-Tregs function, reduced Nrp-1+Tregs frequency, the release of IL-35, IL-10, and TGF-ß1, while increased IFN-γ Teff-secretion. The CXCR4 ligand, CXCL12, recruited CD25+PTEN+Tregs in RCC while R54 significantly reduced it. IL-2/PMA activates Tregs reducing pAKT+Tregs while R54 increases it. The AKT inhibitor, TCB, prevented the increase in pAKT+Tregs R54-mediated. Moreover, R54 significantly reduced FOXP3-TSDR demethylation with DNMT1 and FOXP3 downregulation. CONCLUSION: R54 impairs Tregs function in primary RCC patients targeting PTEN/PI3K/AKT pathway, reducing TSDR demethylation and FOXP3 and DNMT1 expression. Thus, CXCR4 targeting is a strategy to inhibit Tregs activity in the RCC tumour microenvironment.

Hepatocellular carcinoma (HCC) tumor microenvironment is more suppressive than colorectal cancer liver metastasis (CRLM) tumor microenvironment.

BACKGROUND AND PURPOSE: While HCC is an inflammation-associated cancer, CRLM develops on permissive healthy liver microenvironment. To evaluate the immune aspects of these two different environments, peripheral blood-(PB), peritumoral-(PT) and tumoral tissues-(TT) from HCC and CRLM patients were evaluated. METHODS: 40 HCC and 34 CRLM were enrolled and freshly TT, PT and PB were collected at the surgery. PB-, PT- and TT-derived CD4+CD25+ Tregs, M/PMN-MDSC and PB-derived CD4+CD25- T-effector cells (Teffs) were isolated and characterized. Tregs' function was also evaluated in the presence of the CXCR4 inhibitor, peptide-R29, AMD3100 or anti-PD1. RNA was extracted from PB/PT/TT tissues and tested for FOXP3, CXCL12, CXCR4, CCL5, IL-15, CXCL5, Arg-1, N-cad, Vim, CXCL8, TGFß and VEGF-A expression. RESULTS: In HCC/CRLM-PB, higher number of functional Tregs, CD4+CD25hiFOXP3+ was detected, although PB-HCC Tregs exert a more suppressive function as compared to CRLM Tregs. In HCC/CRLM-TT, Tregs were highly represented with activated/ENTPD-1+Tregs prevalent in HCC. As compared to CRLM, HCC overexpressed CXCR4 and N-cadherin/vimentin in a contest rich in arginase and CCL5. Monocytic MDSCs were highly represented in HCC/CRLM, while high polymorphonuclear MDSCs were detected only in HCC. Interestingly, the function of CXCR4-PB-Tregs was impaired in HCC/CRLM by the CXCR4 inhibitor R29. CONCLUSION: In HCC and CRLM, peripheral blood, peritumoral and tumoral tissues Tregs are highly represented and functional. Nevertheless, HCC displays a more immunosuppressive TME due to Tregs, MDSCs, intrinsic tumor features (CXCR4, CCL5, arginase) and the contest in which it develops. As CXCR4 is overexpressed in HCC/CRLM tumor/TME cells, CXCR4 inhibitors may be considered for double hit therapy in liver cancer patients.

CXCR4-CXCL12-CXCR7 and PD-1/PD-L1 in Pancreatic Cancer: CXCL12 Predicts Survival of Radically Resected Patients.

Pancreatic ductal adenocarcinoma (PDAC) is currently the most deadly cancer. Although characterized by 5-20% of neoplastic cells in the highly fibrotic stroma, immunotherapy is not a valid option in PDAC treatment. As CXCR4-CXCL12 regulates tumor invasion and T-cell access and PD-1/PD-L1 controls immune tolerance, 76 PDACs were evaluated for CXCR4-CXCL12-CXCR7 and PD-1/PD-L1 in the epithelial and stromal component. Neoplastic CXCR4 and CXCL12 discriminated PDACs for recurrence-free survival (RFS), while CXCL12 and CXCR7 discriminated patients for cancer-specific survival (CSS). Interestingly, among patients with radical resection (R0), high tumor CXCR4 clustered patients with worse RFS, high CXCL12 identified poor prognostic patients for both RFS and CSS, while stromal lymphocytic-monocytic PD-L1 associated with improved RFS and CSS. PD-1 was only sporadically expressed (<1%) in focal lymphocyte infiltrate and does not impact prognosis. In multivariate analysis, tumoral CXCL12, perineural invasion, and AJCC lymph node status were independent prognostic factors for RFS; tumoral CXCL12, AJCC Stage, and vascular invasion were independent prognostic factors for CSS. CXCL12's poor prognostic meaning was confirmed in an additional perspective-independent 13 fine-needle aspiration cytology advanced stage-PDACs. Thus, CXCR4-CXCL12 evaluation in PDAC identifies prognostic categories and could orient therapeutic approaches.

Tailoring the Structure of Cell Penetrating DNA and RNA Binding Nucleopeptides.

Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these molecules into drugs is a long and difficult process that justifies the continuous research of new chemotypes endowed with favorable binding, pharmacokinetic and pharmacodynamic properties. In this scenario, we describe the synthesis of two sets of homo-thymine nucleopeptides, in which nucleobases are inserted in a peptide structure, to investigate the role of the underivatized amino acid residue and the distance of the nucleobase from the peptide backbone on the nucleic acid recognition process. It is worth noting that the CD spectroscopy investigation showed that two of the reported nucleopeptides, consisting of alternation of thymine functionalized L-Orn and L-Dab and L-Arg as underivatized amino acids, were able to efficiently bind DNA and RNA targets and cross both cell and nuclear membranes.

AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer.

Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/ß pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions.

Biological Role of Tumor/Stromal CXCR4-CXCL12-CXCR7 in MITO16A/MaNGO-OV2 Advanced Ovarian Cancer Patients.

This study investigated the prognostic role of the CXCR4-CXCL12-CXCR7 axis in advanced epithelial ovarian cancer (EOC) patients receiving first-line treatment within the MITO16A/MaNGO-OV2 phase-IV trial. CXCR4-CXCL12-CXCR7 expression was evaluated in the epithelial and stromal component of 308 EOC IHC-stained tumor samples. The statistical analysis focused on biomarkers' expression, their association with other variables and prognostic value. Zero-inflated tests, shrinkage, bootstrap procedures, and multivariable models were applied. The majority of EOC (75.0%) expressed CXCR4 and CXCR7, 56.5% expressed the entire CXCR4-CXCL12-CXCR7 axis, while only 4.6% were negative for CXCL12 and its cognate receptors, in regard to the epithelial component. Stromal CXCL12 and CXCR7, expressed in 11.2% and 65.5%, respectively, were associated with the FIGO stage. High CXCL12 in epithelial cancer cells was associated with shorter progression-free and overall survival. However, after adjusting for overfitting due to best cut-off multiplicity testing, the significance was lost. This is a wide-ranging, prospective study in which CXCR4-CXCL12-CXCR7 were systematically evaluated in epithelial and stromal components, in selected stage III-IV EOC. Although CXCL12 was not prognostic, epithelial expression identified high-risk FIGO stage III patients for PFS. These data suggest that it might be worth studying the CXCL12 axis as a therapeutic target to improve treatment efficacy in EOC patients.

In PD-1+ human colon cancer cells NIVOLUMAB promotes survival and could protect tumor cells from conventional therapies.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent and deadly tumors worldwide. The majority of CRC is resistant to anti-programmed cell death-1 (PD-1)-based cancer immunotherapy, with approximately 15% with high-microsatellite instability, high tumor mutation burden, and intratumoral lymphocytic infiltration. Programmed death-ligand 1 (PD-L1)/PD-1 signaling was described in solid tumor cells. In melanoma, liver, and thyroid cancer cells, intrinsic PD-1 signaling activates oncogenic functions, while in lung cancer cells, it has a tumor suppressor effect. Our work aimed to evaluate the effects of the anti-PD-1 nivolumab (NIVO) on CRC cells. METHODS: In vitro NIVO-treated human colon cancer cells (HT29, HCT116, and LoVo) were evaluated for cell growth, chemo/radiotherapeutic sensitivity, apoptosis, and spheroid growth. Total RNA-seq was assessed in 6-24 hours NIVO-treated human colon cancer cells HT29 and HCT116 as compared with NIVO-treated PES43 human melanoma cells. In vivo mice carrying HT29 xenograft were intraperitoneally treated with NIVO, OXA (oxaliplatin), and NIVO+OXA, and the tumors were characterized for growth, apoptosis, and pERK1/2/pP38. Forty-eight human primary colon cancers were evaluated for PD-1 expression through immunohistochemistry. RESULTS: In PD-1+ human colon cancer cells, intrinsic PD-1 signaling significantly decreased proliferation and promoted apoptosis. On the contrary, NIVO promoted proliferation, reduced apoptosis, and protected PD-1+ cells from chemo/radiotherapy. Transcriptional profile of NIVO-treated HT29 and HCT116 human colon cancer cells revealed downregulation of BATF2, DRAM1, FXYD3, IFIT3, MT-TN, and TNFRSF11A, and upregulation of CLK1, DCAF13, DNAJC2, MTHFD1L, PRPF3, PSMD7, and SCFD1; the opposite regulation was described in NIVO-treated human melanoma PES43 cells. Differentially expressed genes (DEGs) were significantly enriched for interferon pathway, innate immune, cytokine-mediated signaling pathways. In vivo, NIVO promoted HT29 tumor growth, thus reducing OXA efficacy as revealed through significant Ki-67 increase, pERK1/2 and pP38 increase, and apoptotic cell reduction. Eleven out of 48 primary human colon cancer biopsies expressed PD-1 (22.9%). PD-1 expression is significantly associated with lower pT stage. CONCLUSIONS: In PD-1+ human colon cancer cells, NIVO activates tumor survival pathways and could protect tumor cells from conventional therapies.

CXCL12 Signaling in the Tumor Microenvironment.

Tumor microenvironment (TME) is the local environment of tumor, composed of tumor cells and blood vessels, extracellular matrix (ECM), immune cells, and metabolic and signaling molecules. Chemokines and their receptors play a fundamental role in the crosstalk between tumor cells and TME, regulating tumor-related angiogenesis, specific leukocyte infiltration, and activation of the immune response and directly influencing tumor cell growth, invasion, and cancer progression. The chemokine CXCL12 is a homeostatic chemokine that regulates physiological and pathological process such as inflammation, cell proliferation, and specific migration. CXCL12 activates CXCR4 and CXCR7 chemokine receptors, and the entire axis has been shown to be dysregulated in more than 20 different tumors. CXCL12 binding to CXCR4 triggers multiple signal transduction pathways that regulate intracellular calcium flux, chemotaxis, transcription, and cell survival. CXCR7 binds with high-affinity CXCL12 and with lower-affinity CXCL11, which binds also CXCR3. Although CXCR7 acts as a CXCL12 scavenger through ligand internalization and degradation, it transduces the signal mainly through ß-arrestin with a pivotal role in endothelial and neural cells. Recent studies demonstrate that TME rich in CXCL12 leads to resistance to immune checkpoint inhibitors (ICI) therapy and that CXCL12 axis inhibitors sensitize resistant tumors to ICI effect. Thus targeting the CXCL12-mediated axis may control tumor and tumor microenvironment exerting an antitumor dual action. Herein CXCL12 physiology, role in cancer biology and in composite TME, prognostic role, and the relative inhibitors are addressed.

Aflibercept or bevacizumab in combination with FOLFIRI as second-line treatment of mRAS metastatic colorectal cancer patients: the ARBITRATION study protocol.

BACKGROUND: The intensive study of predictive factors has strongly ameliorated the therapeutic flow-chart of metastatic colorectal cancer (mCRC) by allowing the selection of patients who benefit from specific therapies. For instance, in mRAS (mutated RAS) mCRC patients, anti-EGFR drugs (cetuximab and panitumumab) are not recommended; in this group of patients, the use of anti-angiogenic drugs (bevacizumab and aflibercept) is predominant. However, at progression to standard bevacizumab-based first-line chemotherapy, still to date, there are no studies to guide oncologists in the choice of the best second-line anti-angiogenic drug (bevacizumab beyond progression versus aflibercept). METHODS: ARBITRATION is a prospective, observational study assessing efficacy differences between second-line fluorouracil/irinotecan (FOLFIRI)/bevacizumab versus FOLFIRI/aflibercept at progression to fluoropyrimidines, oxaliplatin and bevacizumab in mRAS mCRC patients. A test power of 80%, a median survival of 9 months from second-line treatment start and a hazard ratio of 0.67 between the two schedules were the basis for statistical design. The final sample will be 220 patients (110 per treatment). The significance will be verified with a two-tailed log-rank test with an alpha value of the I-type error of 5%. Time-to-outcome will be described by Kaplan-Meier curves and prognostic factors studied through multivariable analyses based on the Cox model. Secondary objectives include safety, responses' duration and progression-free survival. A translational research will be conducted to measure several angiogenic proteins in patients' serum before starting the therapy in order to evidence any angiogenic factor patterns related to outcome. DISCUSSION: We present a large, prospective, observational study aiming to answer two scientific questions: (1) outcome differences between second-line treatments with FOLFIRI/bevacizumab beyond progression versus FOLFIRI/aflibercept in mRAS mCRC patients, (2) angiogenic factors' patterns that could associate with efficacy and help oncologists to apply best the therapeutic anti-angiogenic strategies. TRIAL REGISTRATION: The ARBITRATION trial (version 0.0, 13 April 2020) has been registered into the clinicaltrials.gov registry on 20 May 2020 with identifier NCT04397601.

Novel Peptide-Based PET Probe for Non-invasive Imaging of C-X-C Chemokine Receptor Type 4 (CXCR4) in Tumors.

The recently reported CXCR4 antagonist 3 (Ac-Arg-Ala-[DCys-Arg-2Nal-His-Pen]-CO2H) was investigated as a molecular scaffold for a CXCR4-targeted positron emission tomography (PET) tracer. Toward this end, 3 was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononanetriacetic acid (NOTA). On the basis of convincing affinity data, both tracers, [68Ga]NOTA analogue ([68Ga]-5) and [68Ga]DOTA analogue ([68Ga]-4), were evaluated for PET imaging in "in vivo" models of CHO-hCXCR4 and Daudi lymphoma cells. PET imaging and biodistribution studies revealed higher CXCR4-specific tumor uptake and high tumor/background ratios for the [68Ga]NOTA analogue ([68Ga]-5) than for the [68Ga]DOTA analogue ([68Ga]-4) in both in vivo models. Moreover, [68Ga]-4 and [68Ga]-5 displayed rapid clearance and very low levels of accumulation in all nontarget tissues but the kidney. Although the high tumor/background ratios observed in the mouse xenograft model could partially derive from the hCXCR4 selectivity of [68Ga]-5, our results encourage its translation into a clinical context as a novel peptide-based tracer for imaging of CXCR4-overexpressing tumors.

New CXCR4 Antagonist Peptide R (Pep R) Improves Standard Therapy in Colorectal Cancer.

The chemokine receptor CXCR4 is overexpressed and functional in colorectal cancer. To investigate the role of CXCR4 antagonism in potentiating colon cancer standard therapy, the new peptide CXCR4 antagonist Peptide R (Pep R) was employed. Human colon cancer HCT116 xenograft-bearing mice were treated with chemotherapeutic agents (CT) 5-Fluorouracil (5FU) and oxaliplatin (OX) or 5FU and radio chemotherapy (RT-CT) in the presence of Pep R. After two weeks, CT plus Pep R reduced by 4-fold the relative tumor volume (RTV) as compared to 2- and 1.6-fold reductions induced, respectively, by CT and Pep R. In vitro Pep R addition to CT/RT-CT impaired HCT116 cell growth and further reduced HCT116 and HT29 clonal capability. Thus, the hypothesis that Pep R could target the epithelial mesenchyme transition (EMT) process was evaluated. While CT decreased ECAD and increased ZEB-1 and CD90 expression, the addition of Pep R restored the pretreatment expression. In HCT116 and HT29 cells, CT/RT-CT induced a population of CD133+CXCR4+ cells, supposedly a stem-resistant cancer cell population, while Pep R reduced it. Taken together, the results showed that targeting CXCR4 ameliorates the effect of treatment in colon cancer through inhibition of cell growth and reversal of EMT treatment-induced markers, supporting further clinical studies.

Targeting CXCR4 potentiates anti-PD-1 efficacy modifying the tumor microenvironment and inhibiting neoplastic PD-1.

BACKGROUND: Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. METHODS: Mice were subcutaneously injected with MC38 (1 × 106) or B16-hCXCR4 (5 × 105). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1-14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. RESULTS: The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 µM) reduced PES43 cells growth while nivolumab (10 µM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2')-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC+ cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. CONCLUSION: Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.

Cetuximab, irinotecan and fluorouracile in fiRst-line treatment of immunologically-selected advanced colorectal cancer patients: the CIFRA study protocol.

BACKGROUND: Combination of chemotherapies (fluoropirimidines, oxaliplatin and irinotecan) with biologic drugs (bevacizumab, panitumumab, cetuximab) have improved clinical responses and survival of metastatic colorectal cancer (mCRC). However, patients' selection thorough the identification of predictive factors still represent a challange. Cetuximab (Erbitux®), a chimeric monoclonal antibody binding to the Epidermal Growth Factor Receptor (EGFR), belongs to the Immunoglobulins (Ig) grade 1 subclass able to elicite both in vitro and in vivo the Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). ADCC is the cytotoxic killing of antibody-coated target cells by immunologic effectors. The effector cells express a receptor for the Fc portion of these antibodies (FcγR); genetic polymorphisms of FcγR modify the binding affinity with the Fc of IgG1. Interestingly, the high-affinity FcγRIIIa V/V is associated with increased ADCC in vitro and in vivo. Thus, ADCC could partially account for cetuximab activity. METHODS/DESIGN: CIFRA is a single arm, open-label, phase II study assessing the activity of cetuximab in combination with irinotecan and fluorouracile in FcγRIIIa V/V patients with KRAS, NRAS, BRAF wild type mCRC. The study is designed with a two-stage Simon model based on a hypothetical higher response rate (+ 10%) of FcγRIIIa V/V patients as compared to previous trials (about 60%) assuming ADCC as one of the possible mechanisms of cetuximab action. The test power is 95%, the alpha value of the I-type error is 5%. With these assumptions the sample for passing the first stage is 14 patients with > 6 responses and the final sample is 34 patients with > 18 responses to draw positive conclusions. Secondary objectives include toxicity, responses' duration, progression-free and overall survival. Furthermore, an associated translational study will assess the patients' cetuximab-mediated ADCC and characterize the tumor microenvironment. DISCUSSION: The CIFRA study will determine whether ADCC contributes to cetuximab activity in mCRC patients selected on an innovative immunological screening. Data from the translational study will support results' interpretation as well as provide new insights in host-tumor interactions and cetuximab activity. TRIAL REGISTRATION: The CIFRA trial (version 0.0, June 21, 2018) has been registered into the NIH-US National Library of Medicine, ClinicalTrials.gov database with the identifier number ( NCT03874062 ).

Ionizing radiation effects on the tumor microenvironment.

The broad use of radiotherapy (RT) in the management of solid human tumors is based on its ability to damage cellular macromolecules, particularly the DNA, effectively inducing growth arrest and cell death locally in irradiated tumor cells. However, bystander effects, such as the transmission of lethal signals between cells via gap junctions or the production of diffusible cytotoxic mediators, can also contribute to the local antineoplastic action of RT. Traditionally, RT has been considered to exert immunosuppressive effects on the host. This idea largely stems from the radiosensitivity of quiescent lymphocytes and on the use of total body irradiation as part of myeloablative conditioning regimens preceding hematopoietic stem cell transplantation. Additionally, the occurrence of the so-called "abscopal effect," where nonirradiated distant lesions display effects of RT response, suggests that RT may also induce tumor immunization. Several RT-induced effects on cancer, immune and stromal cells, contribute to the abscopal effect: (1) induction of "immunogenic cell death", with release of tumor-associated antigens, (2) alterations of cancer cell immunophenotype, and (3) modulation of the tumor microenvironment. Damage and death of cancer cells leads to the surface exposure of immunogenic molecules as well as the release of damage associated molecular patterns such as adenosine triphosphate or High-Mobility-Group-Protein B1, and potentially tumor antigens that activate the innate and adaptive immune systems. Moreover, nuclear release and cytoplasmic sensing of altered nucleic acids via cyclic GMP-AMP Synthase/Stimulator of Interferon Genes is connected to the secretion of cytokines that support innate and adaptive antitumor immunity. As a result of the above, irradiated tumor cells may potentially act as an "in situ vaccine."

CXCL12 loaded-dermal filler captures CXCR4 expressing melanoma circulating tumor cells.

Development of distant metastasis relies on interactions between cancer and stromal cells. CXCL12, also known as stromal-derived factor 1α (SDF-1α), is a major chemokine constitutively secreted in bone marrow, lymph nodes, liver and lung, playing a critical role in the migration and seeding of neoplastic cells. CXCL12 activates the CXCR4 receptor that is overexpressed in several human cancer cells. Recent evidence reveals that tumors induce pre-metastatic niches in target organ producing tumor-derived factors. Pre-metastatic niches represent a tumor growth-favoring microenvironment in absence of cancer cells. A commercially available dermal filler, hyaluronic acid (HA) -based gel, loaded with CXCL12 (CLG) reproduced a "fake" pre-metastatic niche. In vitro, B16-hCXCR4-GFP, human cxcr4 expressing murine melanoma cells efficiently migrated toward CLG. In vivo, CLGs and empty gels (EGs) were subcutaneously injected into C57BL/6 mice and 5 days later B16-hCXCR4-GFP cells were intravenously inoculated. CLGs were able to recruit a significantly higher number of B16-hCXCR4-GFP cells as compared to EGs, with reduced lung metastasis in mice carrying CLG. CLG were infiltrated by higher number of CD45-positive leukocytes, mainly neutrophils CD11b+Ly6G+ cells, myeloid CD11b+Ly6G- and macrophages F4/80. CLG recovered cells recapitulated the features of B16-hCXCR4-GFP (epithelial, melanin rich, MELAN A/ S100/ c-Kit/CXCR4 pos; α-SMA neg). Thus a HA-based dermal filler loaded with CXCL12 can attract and trap CXCR4+tumor cells. The CLG trapped cells can be recovered and biologically characterized. As a corollary, a reduction in CXCR4 dependent lung metastasis was detected.

Expression of AR-V7 and ARv567es in Circulating Tumor Cells Correlates with Outcomes to Taxane Therapy in Men with Metastatic Prostate Cancer Treated in TAXYNERGY.

PURPOSE: Biomarkers aiding treatment optimization in metastatic castration-resistant prostate cancer (mCRPC) are scarce. The presence or absence of androgen receptor (AR) splice variants, AR-V7 and ARv567es, in mCRPC patient circulating tumor cells (CTC) may be associated with taxane treatment outcomes.Experimental Design: A novel digital droplet PCR (ddPCR) assay assessed AR-splice variant expression in CTCs from patients receiving docetaxel or cabazitaxel in TAXYNERGY (NCT01718353). Patient outcomes were examined according to AR-splice variant expression, including prostate-specific antigen (PSA)50 response and progression-free survival (PFS). RESULTS: Of the 54 evaluable patients, 36 (67%) were AR-V7+, 42 (78%) were ARv567es+, 29 (54%) were double positive, and 5 (9%) were double negative. PSA50 response rates at any time were numerically higher for AR-V7- versus AR-V7+ (78% vs. 58%; P = 0.23) and for ARv567es- versus ARv567es+ (92% vs. 57%; P = 0.04) patients. When AR-V mRNA status was correlated with change in nuclear AR from cycle 1 day 1 to day 8 (n = 24), AR-V7+ patients (n = 16) had a 0.4% decrease versus a 12.9% and 26.7% decrease in AR-V7-/ARv567es- (n = 3) and AR-V7-/ARv567es+ (n = 5) patients, respectively, suggesting a dominant role for AR-V7 over ARv567es. Median PFS was 12.02 versus 8.48 months for AR-V7- versus AR-V7+ (HR = 0.38; P = 0.01), and 12.71 versus 7.29 months for ARv567es- versus ARv567es+ (HR = 0.37; P = 0.02). For AR-V7+, AR-V7-/ARv567es+, and AR-V7-/ARv567es- patients, median PFS was 8.48, 11.17, and 16.62 months, respectively (P = 0.0013 for trend). CONCLUSIONS: Although detection of both CTC-specific AR-V7 and ARv567es by ddPCR influenced taxane outcomes, AR-V7 primarily mediated the prognostic impact. The absence of both variants was associated with the best response and PFS with taxane treatment.See related commentary by Dehm et al., p. 1696.

Ligand-Based NMR Study of C-X-C Chemokine Receptor Type 4 (CXCR4)-Ligand Interactions on Living Cancer Cells.

Peptide-binding G protein-coupled receptors (GPCRs) are key effectors in numerous pathological and physiological pathways. The assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we applied multiple ligand-based nuclear magnetic resonance (NMR) methods to study the interaction of peptide heptamers, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) on membranes of human T-Leukemia cells (CCRF-CEM cells). This study represents the first structural investigation reporting the receptor-bound conformation of a peptide to a GPCR directly on a living cell. The results obtained in the field of CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with the CXCR4 field arises. General application of the presented NMR methodologies is possible and surely may help to boost the development of new therapeutic agents targeting GPCRs.

Structure-Activity Relationships and Biological Characterization of a Novel, Potent, and Serum Stable C-X-C Chemokine Receptor Type 4 (CXCR4) Antagonist.

In our ongoing pursuit of CXCR4 antagonists as potential anticancer agents, we recently developed a potent, selective, and plasma stable peptide, Ac-Arg-Ala-[d-Cys-Arg-Phe-Phe-Cys]-COOH (3). Nevertheless, this compound was still not potent enough (IC50 ≈ 53 nM) to enter preclinical studies. Thus, a lead-optimization campaign was here undertaken to further improve the binding affinity of 3 while preserving its selectivity and proteolytic stability. Specifically, extensive structure-activity relationships (SARs) investigations were carried out on both its aromatic and disulfide forming amino acids. One among the synthesized analogue, Ac-Arg-Ala-[d-Cys-Arg-Phe-His-Pen]-COOH (19), displayed subnanomolar affinity toward CXCR4, with a marked selectivity over CXCR3 and CXCR7. NMR and molecular modeling studies disclosed the molecular bases for the binding of 19 to CXCR4 and for its improved potency compared to the lead 3. Finally, biological assays on specific cancer cell lines showed that 19 can impair CXCL12-mediated cell migration and CXCR4 internalization more efficiently than the clinically approved CXCR4 antagonist plerixafor.

Randomized, Noncomparative, Phase II Trial of Early Switch From Docetaxel to Cabazitaxel or Vice Versa, With Integrated Biomarker Analysis, in Men With Chemotherapy-Naïve, Metastatic, Castration-Resistant Prostate Cancer.

Purpose The TAXYNERGY trial ( ClinicalTrials.gov identifier: NCT01718353) evaluated clinical benefit from early taxane switch and circulating tumor cell (CTC) biomarkers to interrogate mechanisms of sensitivity or resistance to taxanes in men with chemotherapy-naïve, metastatic, castration-resistant prostate cancer. Patients and Methods Patients were randomly assigned 2:1 to docetaxel or cabazitaxel. Men who did not achieve ≥ 30% prostate-specific antigen (PSA) decline by cycle 4 (C4) switched taxane. The primary clinical endpoint was confirmed ≥ 50% PSA decline versus historical control (TAX327). The primary biomarker endpoint was analysis of post-treatment CTCs to confirm the hypothesis that clinical response was associated with taxane drug-target engagement, evidenced by decreased percent androgen receptor nuclear localization (%ARNL) and increased microtubule bundling. Results Sixty-three patients were randomly assigned to docetaxel (n = 41) or cabazitaxel (n = 22); 44.4% received prior potent androgen receptor-targeted therapy. Overall, 35 patients (55.6%) had confirmed ≥ 50% PSA responses, exceeding the historical control rate of 45.4% (TAX327). Of 61 treated patients, 33 (54.1%) had ≥ 30% PSA declines by C4 and did not switch taxane, 15 patients (24.6%) who did not achieve ≥ 30% PSA declines by C4 switched taxane, and 13 patients (21.3%) discontinued therapy before or at C4. Of patients switching taxane, 46.7% subsequently achieved ≥ 50% PSA decrease. In 26 CTC-evaluable patients, taxane-induced decrease in %ARNL (cycle 1 day 1 v cycle 1 day 8) was associated with a higher rate of ≥ 50% PSA decrease at C4 ( P = .009). Median composite progression-free survival was 9.1 months (95% CI, 4.9 to 11.7 months); median overall survival was not reached at 14 months. Common grade 3 or 4 adverse events included fatigue (13.1%) and febrile neutropenia (11.5%). Conclusion The early taxane switch strategy was associated with improved PSA response rates versus TAX327. Taxane-induced shifts in %ARNL may serve as an early biomarker of clinical benefit in patients treated with taxanes.

A novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) specifically detects CXCR4 expressing tumors.

C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.