Tumor-penetrating Peptide Research Articles

Coadministration of a tumor-penetrating peptide improves the therapeutic efficacy of paclitaxel in a novel air-grown lung cancer 3D spheroid model.

Three-dimensional (3 D) cell culture platforms are increasingly being used in cancer research and drug development since they mimic avascular tumors in vitro. In this study, we focused on the development of a novel air-grown multicellular spheroid (MCS) model to mimic in vivo tumors for understanding lung cancer biology and improvement in the evaluation of aerosol anticancer therapeutics. 3 D MCS were formed using A549 lung adenocarcinoma cells, comprising cellular heterogeneity with respect to different proliferative and metabolic gradients. The growth kinetics, morphology and 3 D structure of air-grown MCS were characterized by brightfield, fluorescent and scanning electron microscopy. MCS demonstrated a significant decrease in growth when the tumor-penetrating peptide iRGD and paclitaxel (PTX) were coadministered as compared with PTX alone. It was also found that when treated with both iRGD and PTX, A549 MCS exhibited an increase in apoptosis and decrease in clonogenic survival capacity in contrast to PTX treatment alone. This study demonstrated that coadministration of iRGD resulted in the improvement of the tumor penetration ability of PTX in an in vitro A549 3 D MCS model. In addition, this is the first time a high-throughput air-grown lung cancer tumor spheroid model has been developed and evaluated.

Abstract LB-062: Targeting of p32 in peritoneal carcinomatosis with intraperitoneal linTT1 peptide-guided pro-apoptotic nanoparticles

Abstract Gastrointestinal and gynecological malignancies disseminate loco-regionally in the peritoneal cavity - a condition known as peritoneal carcinomatosis (PC). Intraperitoneal (IP) administration can be used to improve therapeutic index of anticancer drugs used for PC treatment. Activity of IP anticancer drugs can be further potentiated by encapsulation in nanocarriers and/or affinity targeting with tumor-specific affinity ligands, such as tumor homing peptides. Here we evaluated a novel tumor penetrating peptide, linTT1 (AKRGARSTA), as a PC targeting ligand for nanoparticles. We first demonstrated that the primary homing receptor for linTT1, p32 (or gC1qR), is expressed on the cell surface of peritoneal carcinoma cell lines of gastric (MKN-45P), ovarian (SKOV-3), and colon (CT-26) origin, and that peritoneal tumors in mice and clinical peritoneal carcinoma explants express p32 protein accessible from the IP space. Iron oxide nanoworms (NWs) functionalized with the linTT1 peptide were taken up and routed to mitochondria in cultured PC cells. NWs functionalized with linTT1 peptide in tandem with a pro-apoptotic [D(KLAKLAK)2] peptide showed p32-dependent cytotoxicity in MKN-45P, SKOV-3, and CT-26 cells. Upon IP administration in mice bearing MKN-45P, SKOV-3, and CT-26 tumors, linTT1-functionalized NWs showed robust homing and penetration into malignant lesions, whereas only a background accumulation was seen in control tissues. In tumors, the linTT1-NW accumulation was seen predominantly in CD31-positive blood vessels, in LYVE-1-positive lymphatic structures, and in CD11b-positive tumor macrophages. Experimental therapy of mice bearing peritoneal MKN-45P xenografts and CT-26 syngeneic tumors with IP linTT1- D(KLAKLAK)2-NWs resulted in significant reduction of weight of peritoneal tumors and significant decrease in the number of metastatic tumor nodules, whereas treatment with untargeted D(KLAKLAK)2-NWs had no effect. Our data show that targeting of p32 with linTT1 tumor-penetrating peptide improves tumor selectivity and antitumor efficacy of IP pro-apoptotic NWs. P32-directed intraperitoneal targeting of other anticancer agents and nanoparticles using peptides and other affinity ligands may represent a general strategy to increase their therapeutic index. Citation Format: Tambet Teesalu, Hunt Hedi, Lorena Simón-Gracia, Allan Tobi, Kazuki N. Sugahara, Shweta Sharma, Erkki Ruoslahti. Targeting of p32 in peritoneal carcinomatosis with intraperitoneal linTT1 peptide-guided pro-apoptotic nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-062. doi:10.1158/1538-7445.AM2017-LB-062

Abstract 5130: Tumor-penetrating peptide-coated nanoparticles as a novel strategy for the targeted therapy of neuroblastoma

Abstract Anticancer drugs loaded into tumor- and vasculature-targeted nanocarriers (NC) can reduce side-effects and improve therapeutic efficacy in pre-clinical studies. However, poorly perfused and dysfunctional tumor vessels and lymphatics limit the transport of the payload into the parenchyma of solid tumors. The use of NC decorated with tumor-penetrating peptides (TPPs) might enhance tumor penetration and antitumor effects. A previously characterized neuroblastoma (NB)-targeting peptide ligand was here modified (now referred as TPP-NB) by adding a consensus motif as a mediator of cell, vascular and tissue penetration via neuropilin-1 (NRP-1) receptor recognition. NPR-1 expression was validated by FACS analysis in NB cell lines and by IHC staining in tumor cells and tumor stroma from NB-bearing mice. Recombinant NRP-1 was used to validate TPP-NB specificity. In vitro and in vivo cell association and internalization of TPP-NB, either free or coupled to Liposomes (L) were tested by FACS and confocal microscopy. Vascular permeability assay after treatment with TPP-NB-targeted, doxorubicin-loaded Liposomes (TPP-NB-L[DXR]) was performed evaluating the in vivo accumulation of Evans Blue dye within the tumor mass. Therapeutic experiments with TPP-NB-L[DXR] were performed in mice orthotopically injected with human NB cells. NRP-1 expression is validated in a panel of NB cells and in tumors from NB-bearing mice. Differently from the original peptide and some control ones, TPP-NB is able to recognize recombinant NRP-1. The addition of the NRP-1-recognizing sequence to the original peptide significantly increases its NB cellular association in vitro. Interestingly, the results seem to indicate that the enhanced capability by TPP-NB in binding NB cells is related to the combination of the NRP-1-recognizing and the original sequence. Importantly, TPP-NB coupled at the external surfaces of L[DXR] significantly increases their cellular association on NB cells in vitro. Competitive binding assay reveals that binding of TPP-NB is specific and can be inhibited by an excess of the unlabeled free peptide. The localization and the cellular distribution of L evaluated by confocal microscopy in vitro and in mouse models of NB, confirm the binding specificity, showing an increased selective internalization of TPP-NB-L-FITC compared to that obtained with either untargeted L or L decorated with the scrambled peptide. Moreover, TPP-NB-L[DXR] further increases the vascular permeability into the NB tumor mass, but not in non-tumor tissues. The therapeutic efficacy of TPP-NB-L[DXR] has been investigating in terms of overall survival. On running results indicate that the novel NC exerts an increased anti-NB effect compared to DXR-loaded L decorated with the original peptide. Our findings demonstrate that the achieved penetrating features by a NB-targeting peptide might increase liposomal drug binding, homing and antitumor efficacy. Citation Format: Fabio Pastorino, Chiara Brignole, Laura Emionite, Silvia Bruno, Flavio Curnis, Daniela Di Paolo, Patrizia Perri, Alessandro Gori, Renato Longhi, Michele Cilli, Angelo Corti, Mirco Ponzoni. Tumor-penetrating peptide-coated nanoparticles as a novel strategy for the targeted therapy of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5130. doi:10.1158/1538-7445.AM2017-5130

Abstract 5088: Tumor penetrating RNA delivery for therapeutic benefit of pancreatic cancer

Abstract Pancreatic cancer remains a deadly disease with a median survival of less than one year, in part because we lack effective therapeutic strategies. Mutations of the oncogene KRAS and the tumor suppressor genes TP53, INK4A, and DPC4 play important roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC); however, these driver events have proven to be challenging targets for drug development. RNA interference through small interfering RNA (siRNA) delivery has the potential to have an impact on silencing ‘undruggable’ targets; however, siRNA delivery in tumors has been challenging in general and worsened in PDAC by the stromal barriers to drug delivery. Herein, we integrated biomedical engineering, materials science, and pre-clinical investigation to establish a novel class of tumor-penetrating nanoparticles (TPNs) to enhance delivery of siRNAs in PDAC for therapeutic benefit. Specifically, we adapted self-assembled nanoparticles comprised of siRNA complexed with tandem tumor-penetrating and membrane-translocating peptides, which enabled the specific delivery of siRNA deep into the tumor parenchyma. Specifically, to establish efficacious siRNA delivery vehicles, we characterized and selected tandem peptides that incorporate novel tumor-penetrating peptide domains known to target PDAC in mice. In parallel, we optimized the particle stability through incorporation of hydrophilic molecules, focusing on achieving desirable pharmacokinetic properties which will reduce off-target delivery and increase potency of a given siRNA dose. The resulting TPNs carry siRNA into the cytosol of monolayer cultures of tumor cells, and also exhibit penetration in pancreas organoids derived from primary mouse and human tumors. As a proof-of-concept, we used TPN in vivo to knockdown KRAS in a murine PDAC model, using subcutaneously-implanted, autochthonous KRASG12D/+p53-/- tumors. Treatment of tumor-bearing mice with systemically administrated KRAS-specific TPNs suppressed the growth of established tumors. With the delivery platform established, the ongoing goals of this project are to identify siRNA targets that synergize with the suppression of oncogenic KRAS, and to validate synthetic lethal targets by interfering with signature PDAC driver events uncovered by functional genomic efforts. Together, these studies will provide a foundation for translational studies involving new targets and delivery technology in pancreatic cancer. Citation Format: Liangliang Hao, Justin Lo, Sangeeta Bhatia. Tumor penetrating RNA delivery for therapeutic benefit of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5088. doi:10.1158/1538-7445.AM2017-5088

Abstract 965: Two in one: nanotechnology based strategies for the treatment of ER+ breast cancer

Abstract Understanding endocrine resistance mechanisms is key to develop new therapeutic strategies. We focus on the role the tumor microenvironment plays as a modulator of endocrine therapy resistance in breast cancer, in particular associated to Tamoxifen. Studies show that the use of nanoparticles (NPs) as antitumor drug delivery systems is a good strategy to improve the efficacy and decrease secondary effects of conventional chemotherapies. In this context we hypothesize that a therapeutic strategy based on the use of Tamoxifen carried in NPs coated with the tumor penetrating peptide iRGD, would be more effective than conventional Tamoxifen. Neuropilin-1, that mediates iRGD induced endocytosis, has been shown to be associated to breast cancer stem cells. Thus, we postulate that our multifunctional NPs would be effective in reducing this cell population, contrary to what is observed with free Tamoxifen. Moreover, iRGD blocks the interaction between integrin β1 and fibronectin, a mechanism we have previously shown induces Tamoxifen resistance (Pontiggia et al. 2012). NPs were synthesized with polyethyleneglycol and polycaprolactone and coated with the iRGD peptide. This peptide was constructed with a FAM fluorophore in order to track the NPs. To evaluate the cell uptake, MCF-7 cells were incubated for 5 h with iRGD-NP or FAM-NP as a control. NP entry was higher when the NPs were coated with iRGD, both in 2D and in 3D cultures. Cell viability experiments revealed that Tamoxifen encapsulated in NPs was more effective than the free drug (p<0,05), and NPs coated with iRGD had an even stronger effect (p<0,01). Moreover, resistance to Tamoxifen induced by fibronectin was reversed with the iRGD-coated NPs, contrary to what is observed with the free drug. Previously, we demonstrated that treatment with Tamoxifen enhances the stem cell population in estrogen receptor-positive breast cancer cell lines (Raffo et al. 2013). In this context iRGD coated NPs lead to a decrease in breast cancer stem cells as evaluated by mammosphere assays (p<0,01). Finally, in vivo tumor homing was evaluated to test whether the iRGD peptide affected the biodistribution of the NPs. Nude mice carrying MCF-7 breast tumors were treated with iRGD-NP and control NPs. Tumors were collected at 4 and 24 hrs post inoculation. Frozen sections were analyzed by confocal microscopy revealing that only the mice treated with the iRGD-NPs had high levels of NPs in the tumor, compared to the control group. Moreover, frozen sections of kidneys, livers, lungs and spleen showed very low levels of iRGD-NPs, confirming that the targeting was effective. In vivo experiments of the impact of iRGD-NPs on tumor growth, metastasis and the stem cell population are ongoing. These results suggest that iRGD-NPs carrying Tamoxifen could be an effective strategy to avoid the development of endocrine resistance in breast cancer. Citation Format: Maria Ines Diaz Bessone, Lorena Simón-Gracia, Pablo Scodeller, Tambet Teesalu, Marina Simian. Two in one: nanotechnology based strategies for the treatment of ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2017-965

Tumor-penetrating Peptide-integrated Thermally Sensitive Liposomal Doxorubicin Enhances Efficacy of Radiofrequency Ablation in Liver Tumors.

Purpose To investigate the role of a tumor-penetrating peptide (internalizing CRGDRGPDC [iRGD])-integrated thermally sensitive liposomal (TSL) doxorubicin (DOX) in combination with radiofrequency (RF) ablation of liver tumors in an animal model. Materials and Methods Approval from the institutional animal care and use committee was obtained. Characterization of iRGD-TSL-DOX was performed in vitro. Next, H22 liver adenocarcinomas were implanted in 138 mice in vivo. The DOX accumulation and cell apoptosis of iRGD-TSL-DOX and TSL-DOX with or without RF were evaluated (n = 5) at different time points after treatment with quantitative analysis or pathologic staining. Mice bearing tumors were randomized into the following six groups (each group, eight mice): no treatment, iRGD-TSL-DOX, TSL-DOX, RF alone, RF ablation followed by TSL-DOX at 30 minutes (TSL-DOX combined with RF), and RF ablation followed by iRGD-TSL-DOX (iRGD-TSL-DOX combined with RF). Kaplan-Meier method was used to estimate the survival curves and log-rank test was used for comparison with statistical software. Results DOX encapsulation efficiency in iRGD-TSL-DOX was 97.5% ± 1.3 (standard deviation) with temperature-dependent drug release capability confirmed in vitro. In vivo, the iRGD-TSL-DOX group had overall higher DOX concentration in the tumor and had maximal difference at 24 hours compared with TSL-DOX group (2.7-fold). RF caused more intense cell apoptosis at 24 hours (median, 65% vs 21%, respectively; P < .001). For end-point survival, the iRGD-TSL-DOX combined with RF group had better survival (median, 32 days) than TSL-DOX combined with RF (median, 27 days; P = .035) or RF alone (median, 21 days; P < .001). Conclusion Conjugation to iRGD helped to improve intratumoral DOX accumulation and further enhanced the activity of TSL-DOX in RF ablation of liver tumors. © RSNA, 2017 Online supplemental material is available for this article.

Targeting of p32 in peritoneal carcinomatosis with intraperitoneal linTT1 peptide-guided pro-apoptotic nanoparticles

Gastrointestinal and gynecological malignancies disseminate in the peritoneal cavity - a condition known as peritoneal carcinomatosis (PC). Intraperitoneal (IP) administration can be used to improve therapeutic index of anticancer drugs used for PC treatment. Activity of IP anticancer drugs can be further potentiated by encapsulation in nanocarriers and/or affinity targeting with tumor-specific affinity ligands, such as tumor homing peptides. Here we evaluated a novel tumor penetrating peptide, linTT1 (AKRGARSTA), as a PC targeting ligand for nanoparticles. We first demonstrated that the primary homing receptor for linTT1, p32 (or gC1qR), is expressed on the cell surface of peritoneal carcinoma cell lines of gastric (MKN-45P), ovarian (SKOV-3), and colon (CT-26) origin, and that peritoneal tumors in mice and clinical peritoneal carcinoma explants express p32 protein accessible from the IP space. Iron oxide nanoworms (NWs) functionalized with the linTT1 peptide were taken up and routed to mitochondria in cultured PC cells. NWs functionalized with linTT1 peptide in tandem with a pro-apoptotic [D(KLAKLAK)2] peptide showed p32-dependent cytotoxicity in MKN-45P, SKOV-3, and CT-26 cells. Upon IP administration in mice bearing MKN-45P, SKOV-3, and CT-26 tumors, linTT1-functionalized NWs showed robust homing and penetration into malignant lesions, whereas only a background accumulation was seen in control tissues. In tumors, the linTT1-NW accumulation was seen predominantly in CD31-positive blood vessels, in LYVE-1-positive lymphatic structures, and in CD11b-positive tumor macrophages. Experimental therapy of mice bearing peritoneal MKN-45P xenografts and CT-26 syngeneic tumors with IP linTT1-D(KLAKLAK)2-NWs resulted in significant reduction of weight of peritoneal tumors and significant decrease in the number of metastatic tumor nodules, whereas treatment with untargeted D(KLAKLAK)2-NWs had no effect. Our data show that targeting of p32 with linTT1 tumor-penetrating peptide improves tumor selectivity and antitumor efficacy of IP pro-apoptotic NWs. P32-directed intraperitoneal targeting of other anticancer agents and nanoparticles using peptides and other affinity ligands may represent a general strategy to increase their therapeutic index.

Tumor-penetrating peptide iRGD conjugation effectively potentiates intratumor T cell infiltration.

e14543 Background: Poor infiltration of activated lymphocytes into solid tumors can be a fundamental factor limiting their efficacy. The purpose of this study was to determine whether a cycled tumor-penetrating peptide, iRGD, with a well-defined role in delivering drugs into extravascular tumor tissues, could facilitate lymphocytes infiltration into tumors in both the iRGD combination regimen and conjugated pattern. Methods: We used polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives, a time-efficient and versatile platform, to immobilize iRGD on T cell membrane, and examined the ability of iRGD to improve lymphocytes infiltration into tumors in multicellular spheroids and two xenograft mouse models. Furthermore, the synergy effect of iRGD modification and PD-1 gene knock out in adoptive T cell transfer immunotherapy was evaluated in a xenograft model of EBV associated gastric cancer. Results: In this study, we first showed that systematic injection of iRGD could double the number of CD3+ T cells in 4T1 tumor in BALB/C mouse and the synthetic iRGD-PEG-lipid, which could spontaneously transfer from solution to cell surfaces, had no effect to cell vitality, proliferation, phenotype and function. In adoptive transfer studies, the infiltration of T cells could be promoted by the co-administration with iRGD while T cells modified with iRGD could spread more extensively throughout both the multicellular spheroids and tumor mass. Furthermore, We demonstrated that iRGD modified T cells had superior antitumor efficiency to iRGD combination regimen due to increased T cell infiltration, and exhibited robust synergy effect with PD-1 gene knock out. Conclusions: Our study offers a simple method to target immune cells into tumors and may have the potential to enhance the clinical efficacy of adoptive T cell immunotherapy.

Tumor-penetrating peptide enhances transcytosis of silicasome-based chemotherapy for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal; however, some improvement in overall survival has been achieved with the introduction of nanocarriers that deliver irinotecan or paclitaxel. Although it is generally assumed that nanocarriers rely principally on abnormal leaky vasculature for tumor access, a transcytosis transport pathway that is regulated by neuropilin-1 (NRP-1) has recently been reported. NRP-1-mediated transport can be triggered by the cyclic tumor-penetrating peptide iRGD. In a KRAS-induced orthotopic PDAC model, coadministration of iRGD enhanced the uptake of an irinotecan-loaded silicasome carrier that comprises lipid bilayer-coated mesoporous silica nanoparticles (MSNPs); this uptake resulted in enhanced survival and markedly reduced metastasis. Further, ultrastructural imaging of the treated tumors revealed that iRGD coadministration induced a vesicular transport pathway that carried Au-labeled silicacomes from the blood vessel lumen to a perinuclear site within cancer cells. iRGD-mediated enhancement of silicasome uptake was also observed in patient-derived xenografts, commensurate with the level of NRP-1 expression on tumor blood vessels. These results demonstrate that iRGD enhances the efficacy of irinotecan-loaded silicasome-based therapy and may be a suitable adjuvant in nanoparticle-based treatments for PDAC.

Access granted: iRGD helps silicasome-encased drugs breach the tumor barrier.

In this issue of the JCI, Liu et al. use irinotecan-loaded nanoparticles to treat pancreatic adenocarcinomas in mice. Encapsulating drugs into nanoparticles has distinct advantages: it can improve the pharmacokinetics of the drug, enhance efficacy, and reduce unwanted side effects. A drawback is that the large size of nanoparticles restricts their access to the tumor interior. Liu and colleagues show that the cyclic tumor-penetrating peptide iRGD, reported to be capable of enhancing tumor penetration by drugs, can overcome this limitation to a substantial degree when administered together with the nanoparticles. Pancreatic adenocarcinoma is a challenging malignancy to treat and in desperate need for improved treatments; therefore, advances like this are most welcome.

INGR-Modified Liposomes for Tumor Vascular Targeting and Tumor Tissue Penetrating Delivery in the Treatment of Glioblastoma.

The tumor vascular barrier and tumor stroma barrier become the two main obstacles in the in vivo delivery of nanomedicines. In this study, to overcome the two barriers, we used iNGR, a tumor-penetrating peptide, to modify the liposomes to increase their accumulation and penetration in tumor tissues. First, iNGR-modified sterically stabilized liposomes (iNGR-SSL) were prepared, which showed vesicle sizes of about 100 nm and narrow size distribution. The uptake of iNGR-SSL by U87MG cells and HUVECs were significantly more than that of unmodified liposome. The in vivo imaging study demonstrated that iNGR modification remarkably increased the accumulation of the liposome in orthotopic tumor tissues of animal model. The immunofluorescence staining analysis proved that iNGR-SSL could penetrate through tumor blood vessels and into the deep tumor tissues. The cytotoxicity of iNGR-modified doxorubicin-loaded liposomes (iNGR-SSL/DOX) on U87MG and HUVECs cells in vitro was significantly enhanced than that of unmodified doxorubicin-loaded liposomes (SSL/DOX). The iNGR-SSL/DOX also showed comparatively (p < 0.05) stronger cytotoxicity on tumor than SSL/DOX, which should be resulted from the increased tumor accumulation and penetration mediated by iNGR. This study proved that iNGR peptide modification might be an effective method to enhance the tumor penetrating ability of liposomes in tumor tissue and their antitumor effect.

Tumor-penetrating peptide fused to a pro-apoptotic peptide facilitates effective gastric cancer therapy.

KLA (sequence, KLAKLAKKLAKLAK) is a peptide which leads to programmed cell death by disrupting the mitochondrial membrane. However, low penetration in tumors greatly limits its application and efficacy. To develop a KLA-based cancer therapy, KLA-iRGD, a recombinant protein was constructed. It consists of the KLA peptide and iRGD(CRGDKGPDC), a tumor-homing peptide with high penetration into tumor tissue and cells. The conjugated KLA exhibits pro-apoptotic activity to prevent the growth of a tumor once it is inside the cell. Once KLA-iRGD is internalized in cultured tumor cells, via the activation of the receptor neuropilin-1, it spreads extensively throughout the mass of the tumor. The recombinant KLA-iRGD protein showed antitumor activity invivo in mice and invitro in tumor cell lines. Repeated treatment with KLA-iRGD greatly prevented tumor growth, resulting in a considerable reduction in tumor volume. According to our data, KLA-iRGD may serve as a potential anticancer agent with limited systemic toxicity and high selectivity for the treatment of MKN45 gastric cancer, which may lead to the enhancement of new targeted anticancer agents.

Replication Study: Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper "Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs" (Sugahara et al., 2010). Here we report the results of those experiments. We found that coadministration with iRGD peptide did not have an impact on permeability of the chemotherapeutic agent doxorubicin (DOX) in a xenograft model of prostate cancer, whereas the original study reported that it increased the penetrance of this cancer drug (Figure 2B; Sugahara et al., 2010). Further, in mice bearing orthotopic 22Rv1 human prostate tumors, we did not find a statistically significant difference in tumor weight for mice treated with DOX and iRGD compared to DOX alone, whereas the original study reported a decrease in tumor weight when DOX was coadministered with iRGD (Figure 2C; Sugahara et al., 2010). In addition, we did not find a statistically significant difference in TUNEL staining in tumor tissue between mice treated with DOX and iRGD compared to DOX alone, while the original study reported an increase in TUNEL positive staining with iRGD coadministration (Figure 2D; Sugahara et al., 2010). Similar to the original study (Supplemental Figure 9A; Sugahara et al., 2010), we did not observe an impact on mouse body weight with DOX and iRGD treatment. Finally, we report meta-analyses for each result.

NRP1 transport of cancer therapeutics mediated by tumor-penetrating peptides

Whereas uptake of low molecular weight agents is generally inhibited in tumors due to high interstitial pressure, tumor uptake of macromolecules is increased due to enhanced permeability and retention (EPR). Small molecule drugs alone or incorporated in nanoparticles (NP) have largely been dependent on such physical tumor uptake (passive) for therapeutic activity. Although passive targeted NP such as Stealth Liposomal Doxorubicin (Doxil ®) are effective with improved safety, drug delivery to tumors is still significantly limited. To improve tumor delivery and efficacy, tumor-penetrating peptides (TPP), which contain sequences that target the tumor and activate the neuropilin-1 receptor (NRP1), have either been co-administered with or conjugated to both small and large therapeutic molecules. In this review, we will discuss TPP-mediated therapeutics which target the NRP1 transport system of tumors.

Inducing Optimal Antitumor Immune Response through Coadministering iRGD with Pirarubicin Loaded Nanostructured Lipid Carriers for Breast Cancer Therapy.

Chemotherapeutic agents trigger antitumor immune response through inducing immunogenic tumor cell death. However, severe toxicity to immune system and insufficient immunogenic cell death hinder chemotherapy from arousing efficient antitumor immunity in vivo. In this study, the cytotoxic drug, pirarubicin (THP), was entrapped into nanostructured lipid carriers (NLC); THP-NLC significantly reduced the toxicity of THP to immune system and improved immune status of breast cancer bearing mice. When THP-NLC was coinjected with iRGD (a tumor-penetrating peptide), drug accumulation in tumors was greatly elevated, which led to significant control of tumor growth and increase of immunogenic tumor cell death. Subsequently, the cytotoxic T lymphocytes (CD3+ and CD8+ cells) infiltration and cytokine (IFN-γ and INF-α) secretion in tumors were heavily increased. The efficient T-cell dependent control of tumors in the late stage and the lower side effects contributed to the longest whole survival of THP-NLC + iRGD treated mice. Therefore, the coadministration of THP-NLC with iRGD resulted in increased tumor cell direct-killing death and enhanced antitumor immune response. Our results illustrated that THP could serve as an immunogenic cell death inducer and the proposed drug delivery strategy might impact cancer immunotherapy by arousing increased immunogenic tumor cell death.

Pharmacokinetic problems in peritoneal drug administration: an update after 20 years.

Intraperitoneal chemotherapy has demonstrated significant pharmacologic and clinical advantage over traditional intravenous administration for cancers that are restricted to the peritoneal cavity. The combination of cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (HIPEC) has become the standard technique used to fight ovarian and gastrointestinal cancers in many centers. However, challenges remain for HIPEC to contact the entire peritoneal surface, penetrate the tumor tissue, and transport to the lymphatics and other metastatic sites. New innovations in delivery technique, such as heated aerosol, and in delivery molecules, such as microparticles, nanoparticles, nanogels, and tumor-penetrating peptides are being tested in animal models and will likely soon be in human trials. Improvements in overall care, such as the recent clinical trial of an oral agent for maintenance therapy in ovarian carcinoma, will continue in this field for the next 20 years.

Abstract IA12: Stromal cells in the targeting and transport of tumor-penetrating peptides

Abstract This laboratory studies tissue-specific vascular markers, “vascular zip codes”, and their use in selective targeting of drugs to diseased tissues. Screening of phage libraries in live mice identifies peptides that direct phage homing to a specific target in the body. When the libraries are injected into the circulation, tissue-specific or tumor-specific differences in endothelial cells are primarily targeted. The endothelial target molecules are frequently also expressed by other tumor stromal cells and tumor cells, but not cells in normal tissues. Recently discovered tumor-penetrating peptides, through a complex mechanism that involves a proteolytic cleavage and binding to a second receptor, activate an endocytic transport pathway related to but distinct from macropinocytosis. This trans-tissue pathway, dubbed the CendR pathway, mediates the exit of payloads ranging from small molecule drugs to nanoparticles from the blood vessels and transport through extravascular tumor tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a drug, antibody, or nanoparticle payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Remarkably the payload does not have to be coupled to the peptide; the peptide activates a bulk transport system that sweeps along any compound that is present in the blood. Treatment studies in mice show improved anti-tumor efficacy and less damage to normal tissues. Citation Format: Erkki Ruoslahti, Erkki Ruoslahti. Stromal cells in the targeting and transport of tumor-penetrating peptides. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA12.

262. Therapeutic Suppression of the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts with U1 Adaptor Oligonucleotide / RGD Peptide Conjugates

U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein (U1 snRNP) complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA inside the nucleus. In contrast to siRNA or antisense oligos, U1 Adaptors can accept extensive covalent modifications for nuclease resistance, targeted delivery or in-vivo imaging without loss of silencing activity, offering important advantages as therapeutic agents. A panel of candidate U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MIA-PaCa2. The best candidates reduced KRAS mRNA expression by up to 76% - as effectively as siRNA controls. Reduced KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro and increased apoptosis were seen for both the MIA-PaCa2 (KRASG12C) and Panc1(KRASG12D) cell lines, but not in BxPC3, a KRASwildtype pancreatic cancer cell line. In a parallel project, U1 Adaptors targeting human MYC mRNA were designed and screened in B-cell lymphoma lines, where the best candidates reduced MYC mRNA levels by over 95%. Because of the observed relationship between activating KRAS mutation and MYC overexpression in pancreatic cancers, MYC Adaptors were tested in MIA-PaCa2 cells. MYC Adaptors also inhibited cell growth and increased apoptosis in vitro. U1 Adaptors were tested for efficacy in mice bearing subcutaneous MIA-PaCa2 xenograft tumors. For in-vivo delivery, Adaptor oligos were directly conjugated to a cyclic RGD-motif peptide (cRGD), which is a targeting ligand for specific integrin-family receptors overexpressed on parenchyma and endothelial cells of many solid tumors. Alternately, U1 Adaptor oligos were linked to “internalizing” RGD (iRGD), a variant RGD peptide that also triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin-1. KRAS Adaptors linked to cRGD or iRGD were administered by tail vein injections twice weekly for three to four weeks. Over a series of experiments, tumor growth was inhibited by averages of 68% to 93%. Tumor stasis or regression occurred in some treated mice. In a pilot study, MYC Adaptors conjugated to iRGD peptide were also highly effective in suppressing tumor growth and inducing tumor regression. Excised tumors were analyzed by qPCR and western blot, which confirmed reductions of the targeted mRNAs and proteins. We have shown that U1 Adaptors conjugated to tumor-targeting / tumor-penetrating peptides can effectively target human KRAS and MYC oncogenes in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, MYC and possibly other oncogene targets in pancreatic cancer.

Urokinase-controlled tumor penetrating peptide

Tumor penetrating peptides contain a cryptic (R/K)XX(R/K) CendR element that must be C-terminally exposed to trigger neuropilin-1 (NRP-1) binding, cellular internalization and malignant tissue penetration. The specific proteases that are involved in processing of tumor penetrating peptides identified using phage display are not known. Here we design de novo a tumor-penetrating peptide based on consensus cleavage motif of urokinase-type plasminogen activator (uPA). We expressed the peptide, uCendR (RPARSGR↓SAGGSVA, ↓ shows cleavage site), on phage or coated it onto silver nanoparticles and showed that it is cleaved by uPA, and that the cleavage triggers binding to recombinant NRP-1 and to NPR-1-expressing cells. Upon systemic administration to mice bearing uPA-overexpressing breast tumors, FAM-labeled uCendR peptide and uCendR-coated nanoparticles preferentially accumulated in tumor tissue. We also show that uCendR phage internalization into cultured cancer cells and its penetration in explants of murine tumors and clinical tumor explants can be potentiated by combining the uCendR peptide with tumor-homing module, CRGDC.Our work demonstrates the feasibility of designing tumor-penetrating peptides that are activated by a specific tumor protease. As upregulation of protease expression is one of the hallmarks of cancer, and numerous tumor proteases have substrate specificities compatible with proteolytic unmasking of cryptic CendR motifs, the strategy described here may provide a generic approach for designing proteolytically-actuated peptides for tumor-penetrative payload delivery.

Tumor-Penetrating Peptide-Modified DNA Tetrahedron for Targeting Drug Delivery.

DNA self-assembling nanostructure has been considered as a promising candidate as a drug delivery vehicle because of its compactness, mechanical stability, and noncytotoxicity. In this work, we developed functional, multiform DNA nanostructures by appending a tumor-penetrating peptide to tetrahedral DNA nanostructure (p-TDN). This functional structure is able to efficiently increase the rate of uptake of glioblastoma cell U87MG compared with the DNA tetrahedron and the double-stranded DNA structures. We found that the DNA tetrahedron plays the main role in the endocytosis of U87MG cells, whereas the tumor-penetrating peptide could also bind to transmembrane glycoprotein neuropilin-1 and mediate the endocytosis of the p-TDN nanostructure. Moreover, given the high efficiency of the growth inhibitory effect of the p-TDN loading doxorubicin hydrochloride, the p-TDN distinguishes itself as a promising candidate as an effective delivery carrier.