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Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery., Drugs that sensitize tumour cells to ionizing radiation are prized because they can overcome resistance to radiotherapy. Here, the authors show that anti-tubulin drugs conjugated to cetuximab or trastuzumab can radiosensitize EGFR- or HER2-expressing tumors by increasing DNA damage and cell death due to ionizing radiation.

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP–cRGD–MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP–cRGD–MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. Cancer Res; 75(7); 1376–87. ©2015 AACR.

OBJECTIVE:Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. METHODS:K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPPNleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPPNleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. RESULTS:In chronic arthritis, RACPPNleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPPNleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPPNleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. CONCLUSION:RACPPNleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPPNleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression.

Activatable cell-penetrating peptides (ACPP) provide a general strategy for molecular targeting by exploiting the extracellular protease activities associated with disease. Previous work used a matrix metalloproteinase (MMP-2 and 9)-cleavable sequence in the ACPP to target contrast agents for tumor imaging and fluorescence-guided surgery. To improve specificity and sensitivity for MMP-2, an integrin αvβ3-binding domain, cyclic-RGD, was covalently linked to the ACPP. This co-targeting strategy relies on the interaction of MMP-2 with integrin αvβ3, which are known to associate via the hemopexin domain of MMP-2. In U87MG glioblastoma cells in culture, dual targeting greatly improved ACPP uptake compared with either MMP or integrin αvβ3 targeting alone. In vivo, dual-targeted ACPP treatment resulted in tumor contrast of 7.8 ± 1.6, a 10-fold higher tumor fluorescence compared with the negative control peptide, and increased probe penetration into the core of MDA-MB-231 tumors. This platform also significantly improved efficacy of the chemotherapeutic monomethylauristatin E (MMAE) in both MDA-MB-231 orthotopic human and syngeneic Py230 murine breast tumors. Treatment with cyclic-RGD-PLGC(Me)AG-MMAE-ACPP resulted in complete tumor regression in one quarter of MDA-MB-231 tumor–bearing mice, compared with no survival in the control groups. This rational mechanism for amplified delivery of imaging and potent chemotherapeutic agents avoids the use of antibodies and may be of considerable generality. Mol Cancer Ther; 13(6); 1514–25. ©2014 AACR.

Critical aggregation concentration (CAC) of surfactants is lowered, when polyelectrolytes act as counterions. At a concentration in between the CACs of the surfactant and the polymer-surfactant complex, protein-induced disassemblies can be achieved. This is because, when proteins competitively bind to the polyelectrolytes, the surfactants are not capable of sustaining a micelle-type assembly at this concentration. Since these amphiphilic aggregates are capable of non-covalently sequestering hydrophobic guest molecules, the protein binding induced disassembly process also results in a guest release from these assemblies. We show here that the change in fluorescence with different proteins is not only dependent on the nature of the polymer-surfactant complex, but also on the fluorescent transducer. Two processes can be responsible for the observed fluorescence change: fluorophore guest release from the hydrophobic interior of the assembly or excited state quenching due to complementary components in the analyte. The latter mechanism is especially possible with metalloproteins. We show here that at nanomolar concentration of the proteins, an excited state quenching is possible while at micromolar concentrations the disassembly-based fluorescence reduction is the dominant pathway.

Supramolecular complex of a cationic surfactant and oppositely charged disulfide containing polyelectrolyte was found to form micelle type aggregates at concentration much lower than the critical aggregate concentration (CAC) of the surfactant itself. We show that this difference can be utilized to generate stimulus-sensitive disassembly of these structures. This can be achieved either by converting the polyelectrolyte counterions to monovalent counterions in response to a stimulus or by simply weakening the interaction between the polymer and the surfactant in the presence of a stimulus. We have utilized three different stimuli to demonstrate these possibilities.

Molecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC.Thirteen BRAFV600E mice with PTC were studied-seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy.The average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/- 0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals.RACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone.

Head and neck squamous cell carcinoma (HNSCC) is a source of significant morbidity and mortality worldwide1. Well-established risk factors for HNSCC include tobacco, alcohol, and human papilloma virus (HPV)1. Current staging and clinical management HNSCC patients are based on anatomical location and size2. If patients undergo surgical excision, management decisions are guided by presence or absence of bony invasion, lymph-node metastasis, or lymph-node extracapsular spread. Patients with locally advanced disease are treated with multimodality therapy including chemotherapy, radiotherapy and surgery. Many prevalent malignancies such as breast, colon, lung, or prostate cancer have diagnostic assays based on genomic profiling, HER-2 and estrogen receptor expression, or ALK, EGFR and KRAS mutation. HPV has recently been identified as being responsible for the increasing incidence of oropharyngeal SCC (OPSCC)3,4, and HPV positive (HPV+) tumor status is currently the strongest prognostic factor for these patients4,5. However, there are currently no widely adopted prognostic assays for HPV-negative (HPV−) HNSCC. We previously analyzed The Cancer Genomic Atlas (TCGA), which contains the largest collection of patient HNSCC tumor specimens6. In HPV− tumors, reduced survival outcomes associated with tumor protein p53 (TP53) mutation occur only in combination with loss of chromosome 3p6. In these patients, median survival decreased from >5 years for TP53 mutations to only 1.7 years for both mutational events (“double-hit”)6. These findings are independent of clinical stage and replicated in an independent HNSCC cohort6. In HPV+ tumors, we believe E6-mediated inactivation of TP53 also acts in synergy with 3p deletion to produce poor survival outcomes6. Our prior analysis of the TCGA demonstrated that of the genes on the 3p chromosome, FHIT, a known tumor suppressor gene involved in aerodigestive cancer, best correlates with 3p status6. The FHIT gene is located on a chromosomal fragile site, and loss of the 3p arm is most commonly mediated by chromosome breakage at this site7. Thus, 3p deletion is commonly associated with decreased FHIT protein expression or reduced copy number at the 3p.14.2 FHIT locus7. Given these findings, our analysis in this study uses FHIT copy number as representative of 3p status and thus a proxy for “single hit” (TP53 only) vs “double hit” (TP53 and FHIT deletion) in HNSCC cell lines. It has also been shown that FHIT and p53 status are linked in development of lung cancer, although little is known about the molecular nature of this interaction8. Here, we analyze data from the TCGA to examine whether TP53 and 3p status are linked in HNSCC patients. The etiology for worse clinical outcomes in TCGA patients with “double-hit” cancers is unclear. Potential explanations include greater resistance to radiation monotherapy or more aggressive tumor biology in cancers with the “double hit.” We do not anticipate a difference in cisplatin sensitivity given its lack of efficacy as monotherapy in clinical practice. To examine these hypotheses, we obtained four human tongue squamous cell carcinoma cell lines from American Type Culture Collection (ATCC). We utilized the Cancer Cell Line Encyclopedia (CCLE) to examine differences in their TP53 mutational and FHIT copy number status, corresponding with the prognostic markers identified in the TCGA. To compare radiosensitivity in-vitro, we utilized a comet-tail assay to measure each cell line’s ability to repair ionizing radiation-induced double-stranded deoxyribonucleotide (DNA) damage. FHIT knockout has been previously associated with increased activation of the ATR/CHK1 pathway and stronger cell-cycle checkpoint responses, resulting in greater cellular repair in response to ionizing radiation9. We would thus expect reduced radiation-induced DNA damage in “double-hit” lines with FHIT deletion compared to “single-hit” lines with intact FHIT. We also examined the aggressiveness of “single-hit” versus “double-hit” cell lines by comparing their respective matrix-metalloproteinase (MMP) activity levels. MMP expression has previously been associated with tumor aggressiveness and patient prognosis in head and neck cancer15–19. Absolute levels of MMP2/9 have been used to differentiate between benign papillomas and carcinoma of the larynx15. Increased MMP2/9 expression has been correlated with higher cancer grade16 and reduced survival in HNSCC17–18. In tongue SCC, increased MMP2/9 expression directly correlates with incidence of lymph node metastases19. Recently, our own work has demonstrated that MMP over-expression in HPV+ tumors predicts poorer survival14. Additionally, HPV− tumors, which have a more aggressive phenotype compared to their HPV+ counterparts, also express higher levels of MMP14. We compared MMP activity in our cell lines using previously described ratiometric activatable cell-penetrating peptides (RACPP)10–12. The RACPP relies on tumor-associated MMP2/9 to cleave an intervening linker, resulting in increased Cy5/Cy7 fluorescent signal ratio, and has previously been used for margin detection during surgery12–14.

It has been shown by us in a recent communication that homopolymers, in which each repeat unit contains a hydrophilic and a hydrophobic head group, are capable of forming environment-dependent micellar or inverse micellar assemblies. A systematic structure-property relationship study is carried out here to test the scope of the design. We show here that the molecular design is indeed broadly applicable and that there is a significant gain in the critical aggregation concentrations of these polymers, as compared to the small molecule counterparts. We also show that the design can be tuned to achieve vesicle-type assemblies, which further expands the repertoire of amphiphilic homopolymers in a variety of areas. Characterizations of these assemblies have been carried out using transmission electron microscopy, dynamic light scattering, static light scattering, and dye incorporation experiments.

Objective(1) Obtain matrix-metalloproteinase (MMP) expression profiles for head and neck squamous cell carcinoma (HNSCC) specimens from the Cancer Genomic Atlas (TCGA). (2) Demonstrate HNSCC imaging using MMP-cleavable, fluorescently labeled ratiometric activatable cell-penetrating peptide (RACPP).Study designRetrospective human cohort study; prospective animal study.SettingTranslational research laboratory.Subjects and methodsPatient clinical data and mRNA expression levels of MMP genes were downloaded from TCGA data portal. RACPP provides complementary ratiometric fluorescent contrast (increased Cy5 and decreased Cy7 intensities) when cleaved by MMP2/9. HNSCC-tumor bearing mice were imaged in vivo after RACPP injection. Histology was evaluated by a pathologist blinded to experimental conditions. Zymography confirmed MMP-2/9 activity in xenografts. RACPP was applied to homogenized human HNSCC specimens, and ratiometric fluorescent signal was measured on a microplate reader for ex vivo analysis.ResultsExpression of multiple MMPs including MMP2/9 is greater in patient HNSCC tumors than matched control tissue. In patients with human papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14 expression correlates with worse 5-year survival. Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2/9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Fluorescence ratios were greater in areas of higher tumor burden (P < .03), which is useful for intraoperative margin assessment. Ex vivo, human HNSCC specimens showed greater cleavage of RACPP when compared to control tissue (P = .009).ConclusionsHuman HNSCC tumors show increased mRNA expression of multiple MMPs including MMP2/9. We used RACPP, a ratiometric fluorescence assay of MMP2/9 activity, to show improved occult tumor identification and margin clearance. Ex vivo assays using RACPP in biopsy specimens may identify patients who will benefit from intraoperative RACPP use.

BackgroundThe aim of this study was to evaluate the efficacy of using matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)-cleavable ratiometric activatable cell-penetrating peptides (RACPPs) conjugated to Cy5 and Cy7 fluorophores to accurately label pancreatic cancer for fluorescence-guided surgery (FGS) in an orthotopic mouse model.MethodsOrthotopic mouse models were established using MiaPaCa-2-GFP human pancreatic cancer cells. Two weeks after implantation, tumor-bearing mice were randomized to conventional white light reflectance (WLR) surgery or FGS. FGS was performed at far-red and infrared wavelengths with a customized fluorescence-dissecting microscope 2 h after injection of MMP-2 and MMP-9-cleavable RACPPs. Green fluorescence imaging of the GFP-labeled cancer cells was used to assess the effectiveness of surgical resection and monitor recurrence. At 8 weeks, mice were sacrificed to evaluate tumor burden and metastases.ResultsMice in the WLR group had larger primary tumors than mice in the FGS group at termination [1.72 g ± standard error (SE) 0.58 vs. 0.25 g ± SE 0.14; respectively, p = 0.026). Mean disease-free survival was significantly lengthened from 5.33 weeks in the WLR group to 7.38 weeks in the FGS group (p = 0.02). Recurrence rates were lower in the FGS group than in the WLR group (38 vs. 73 %; p = 0.049). This translated into lower local and distant recurrence rates for FGS compared to WLR (31 vs. 67 for local recurrence, respectively, and 25 vs. 60 % for distant recurrence, respectively). Metastatic tumor burden was significantly greater in the WLR group than in the FGS group (96.92 mm(2) ± SE 52.03 vs. 2.20 mm(2) ± SE 1.43; respectively, χ (2) = 5.455; p = 0.02).ConclusionsRACPPs can accurately and effectively label pancreatic cancer for effective FGS, resulting in better postresection outcomes than for WLR surgery.

Nonconjugated fluorogenic amphiphilic polymers containing an anthracene chromophore exhibit fluorescence quenching in the presence of metalloproteins, although the binding of the polymer to proteins is not selective. The reason for this difference is that the possible conformational changes that protein binding could bring about on a polymer do not affect the fluorescence properties of a pendent chromophore in nonconjugated polymers. This is in contrast to the nonspecific binding and response found with conjugated polymers to proteins.

This review highlights the developments in dendrimer-based micelles for drug delivery. Dendrimers, the perfectly branched monodisperse macromolecules, have certain structural advantages that make them attractive candidates as drug carriers for controlled release or targeted delivery. As polymeric micelle-based approaches precede the work in dendrimers, these are also discussed briefly. The review concludes with a perspective on possible applications of biaryl-based dendrimeric micelles that exhibit environment-dependent conformations, in drug delivery. Keywords: Micelle; drug delivery; dendrimers; controlled release; targeted delivery; polymer−drug conjugates