Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment.

BACKGROUND: Broader clinical acceptance of photodynamic therapy is currently hindered by (a) poor depth efficacy, and (b) predisposition towards establishment of an angiogenic environment during the treatment. Improved depth efficacy is being sought by exploiting the NIR tissue transparency window and by photo-activation using two-photon absorption (2PA). Here, we use two-photon activation of PDT sensitizers, untargeted and targeted to SST2 receptors or EGF receptors, to achieve deep tissue treatment. METHODS: Human tumor lines, positive or negative for SST2r expression were used, as well as murine 3LL cells and bovine aortic endothelial cells. Expression of SST2 receptors on cancer cells and tumor vasculature was evaluated in vitro and frozen xenograft sections. PDT effects on tumor blood flow were followed using in vivo scanning after intravenous injection of FITC conjugated dextran 150K. Dependence of the PDT efficacy on the laser pulse duration was evaluated. Effectiveness of targeting to vascular SST2 receptors was compared to that of EGF receptors, or no targeting. RESULTS: Tumor vasculature stained for SST2 receptors even in tumors from SST2 receptor negative cell lines, and SST2r targeted PDT led to tumor vascular shutdown. Stretching the pulse from ~120fs to ~3ps led to loss of the PDT efficacy especially at greater depth. PDT targeted to SST2 receptors was much more effective than untargeted PDT or PDT targeted to EGF receptors. GENERAL SIGNIFICANCE: The use of octreotate to target SST2 receptors expressed on tumor vessels is an excellent approach to PDT with few recurrences and some long term cures.

Novel guanide-substituted compounds bind to CXCR4 and inhibit breast cancer metastasis.

CXCR4 has been shown to be overexpressed on breast cancer cells including the human MDA-MB-231 cell line. Cancer cells overexpressing the CXCR4 receptor are capable of undergoing metastasis to organs expressing high levels of CXCL12. We have synthesized numerous guanide, biguanide, phenylguanide, and naphthylguanide compounds that bind to CXCR4 at the CXCL12-binding site and thus should prevent CXCR4-facilitated cancer metastasis. The novel compounds presented here were tested for CXCR4 affinity, toxicity, receptor activation, and for their ability to prevent breast cancer metastases. Three of the compounds bound to CXCR4 at IC50 values of 0.06-0.2 µmol/l, with no associated cell toxicity or receptor activation at these concentrations. These high CXCR4 affinity compounds also showed inhibition of in-vitro wound migration. They were then tested in an in-vivo mouse breast cancer lung colony model. All of these compounds showed reductions in the number of MDA-MB-231 lung metastases compared with mock-treated control mice without evidence of cardiac, liver, or kidney toxicity in treated mice.

New two-photon activated photodynamic therapy sensitizers induce xenograft tumor regressions after near-IR laser treatment through the body of the host mouse.

PURPOSE: The aim of this study was to show that novel photodynamic therapy (PDT) sensitizers can be activated by two-photon absorption in the near-IR region of the spectrum and to show, for the first time, that such activation can lead to tumor regressions at significant tissue depth. These experiments also evaluated effects of high-energy femtosecond pulsed laser irradiation on normal tissues and characterized the response of xenograft tumors to our PDT protocols. EXPERIMENTAL DESIGN: Human small cell lung cancer (NCI-H69), non-small cell lung cancer (A549), and breast cancer (MDA-MB-231) xenografts were induced in SCID mice. Irradiation of sensitized tumors was undertaken through the bodies of tumor-bearing mice to give a treatment depth of 2 cm. Posttreatment tumor regressions and histopathology were carried out to determine the nature of the response to these new PDT agents. Microarray expression profiles were conducted to assess the similarity of responses to single and two-photon activated PDT. RESULTS: Regressions of all tumor types tested were seen. Histopathology was consistent with known PDT effects, and no, or minimal, changes were noted in irradiated normal tissues. Cluster analysis of microarray expression profiling showed reproducible changes in transcripts associated with apoptosis, stress, oxygen transport, and gene regulation. CONCLUSIONS: These new PDT sensitizers can be used at a depth of 2 cm to produce excellent xenograft regressions. The tumor response was consistent with known responses to single-photon activated PDT. Experiments in larger animals are warranted to determine the maximal achievable depth of treatment.

Highly sensitive detection of cancer cells using femtosecond dual-wavelength near-IR two-photon imaging.

We describe novel imaging protocols that allow detection of small cancer cell colonies deep inside tissue phantoms with high sensitivity and specificity. We compare fluorescence excited in Styryl-9M molecules by femtosecond pulses at near IR wavelengths, where Styryl-9M shows the largest dependence of the two-photon absorption (2PA) cross section on the local environment. We show that by calculating the normalized ratio of the two-photon excited fluorescence (2PEF) intensity at 1200 nm and 1100 nm excitation wavelengths we can achieve high sensitivity and specificity for determining the location of cancer cells surrounded by normal cells. The 2PEF results showed a positive correlation with the levels of MDR1 proteins expressed by the cells, and, for high MDR1 expressors, as few as ten cancer cells could be detected. Similar high sensitivity is also demonstrated for tumor colonies induced in mouse external ears. This technique could be useful in early cancer detection, and, perhaps, also in monitoring dormant cancer deposits.

Tumor shedding of laminin binding protein modulates angiostatin production in vitro and interferes with plasmin-derived inhibition of angiogenesis in aortic ring cultures.

The growth of solid tumors is largely controlled by the process of angiogenesis. A 67 kDa protein, the laminin binding protein (LBP), is shed from malignant cells in significant amounts and binds to laminin-1 (Starkey et al., Cytometry 1999;35:37-47; Karpatová et al., J Cell Biochem 1996;60:226-34). However, the functions of shed LBP are not fully understood. We hypothesize that matrix-bound LBP could modulate local tumor angiogenesis. In support of this hypothesis, we demonstrate that shed LBP exhibits sulfhydryl oxidase-like activities, and modifies the production of angiostatins from plasmin in vitro. The molecular weights of the autocatalytic products of lys-plasmin incubated with LBP in vitro suggest that PMDs (plasmin A chains attached to degraded B chains) (Ohyama et al., Eur J Biochem 2004;271:809-20) are preferentially generated. Using rat aortic ring assays, we also show that shed LBP reverses plasmin-dependent inhibition of vascular outgrowth. To elucidate which LBP region(s) are active in modulating angiogenesis, limited proteolysis experiments were conducted to determine stable rLBP domains likely to fold correctly, and these were cloned, expressed and purified. The stable LBP fragments were tested for binding to laminin-1 and for competition with shed LBP activity in the aortic ring assay. Results of these studies suggest that the active LBP domains lie within the 137-230 amino acid sequence, a region known to contain 2 bioactive sequences. Since this fragment binds to laminin-1 and modulates angiogenesis, it appears likely that binding of shed LBP to matrix laminin-1 is related to its functions in tumor angiogenesis. The findings presented in this manuscript suggest that LBP shedding could provide a useful therapeutic target.

Comparative modeling of the N-terminal domain of the 67kDa laminin-binding protein: implications for putative ribosomal function.

Laminin-binding protein/p40 (LBP/p40) precursor appears to be involved in two seemingly unrelated activities-cell adhesion and ribosomal biogenesis. Analysis of primary structure revealed a two-domain organization of the LBP/p40. The N-terminal portion of LBP is similar to the S2 family of prokaryotic ribosomal proteins, while the C-terminus is unique for Metazoa and is involved in extraribosomal functions. To gain insight into putative ribosomal functions of LBP we performed comparative modeling of the N-terminal domain using crystal structures of S2p from Thermus thermophilus. The LBP model assumes an alpha-beta sandwich fold similar to that of S2. Modeling revealed the loss of a significant portion of ribosomal RNA (rRNA) interaction domain, lack of conservation of many residues involved in interactions with rRNA, and a major shift in surface charge distribution (compared to the S2 protein). The overall stability of the fold argues against a proposed transmembrane domain in the central part of the protein. Partial overlap in S2 and laminin-binding domains suggests that ribosomal and surface receptor functions would be mutually exclusive. The possible biological role of LBP/p40 bifunctionality is discussed.