Tumor growth and metastasis are angiogenesis-dependent. The possibility of inhibiting tumor growth by interfering with the formation of new vessels has recently raised considerable interest. We previously reported that it is possible to inhibit primary tumor growth and metastasis in a transgenic model of spontaneous breast tumor, which shows many similarities to its human counterpart (including ability to metastasize) by intratumoral administration of a DNA conKeywords: gene therapy; breast adenocarcinoma; metastasis; transgenic mice; anti-angiogenesis; liposomesIt is now widely recognized that the development of new blood vessels (angiogenesis) is necessary to sustain tumor growth, invasion and metastasis. At some point during tumor growth, cancer cells acquire the ability to activate the quiescent vasculature to produce new blood vessels via a so-called 'angiogenic switch '. 1,2 Although the need for recruiting new blood vessels to the tumor has been know for more than two decades, the possibility of treating malignancies by acting on their vasculature has become popular only since the isolation of endogenous angiogenesis inhibitors, which dramatically affect cancer growth in experimental murine systems. Highly promising results on experimental tumors in rodents using these inhibitors as purified proteins as well as transduced genes, alone or in combination with traditional therapies have been reported. [3][4][5][6][7] The establishment of a dormancy status, which in some cases persists even after the suspension of therapy, has been obtained with these gene products, 5 and this, together with the absence of drug resistance, 8 raised the possibility that the anti-angiogenic effects could be useful in the treatment of human tumors.The achievement of the inhibition of local tumor growth is the usual end-point of these therapeutical attempts, but in humans it is the metastatic spread that is responsible for the majority of cancer-related deaths. Therefore, to be really useful in the human context, any new approach must be evaluated in its ability to interfere Correspondence: MG Sacco, ITBA CNR, Via F lli Cervi, 93, 20090 Segrate (MI), Italy Received 17 August 2000; accepted 11 October 2000 struct carrying the murine angiostatin cDNA driven by liposomes. Here we report that it is also possible to achieve this goal by a systemic (intraperitoneal) delivery of therapeutic DNA constructs carrying genes coding for mouse and human anti-angiogenic factors which include angiostatin, endostatin and TIMP-2. These findings may be relevant to the design of therapeutic interventions in humans. Gene Therapy (2001) 8, 67-70. with tumor cell invasion and metastasis, a therapeutical target which is rarely, if ever, investigated at the experimental level. Unfortunately, most of the results in experimental oncology have been obtained on transplantable tumors which are usually generated by inoculating highly malignant cultured cells, which rapidly grow in the site of injection and become the target for therapeutic intervention. ...

Treatments available to women with locally advanced breast cancer are unsatisfactory, since most patients succumb to metastatic spread. Therefore, there is a need to devise novel therapeutic combinations that effectively inhibit metastatization and to test them in animal models of breast cancer showing strong similarities with their human counterpart, including the ability to give rise to metastases. With these considerations in mind, tamoxifen (TAM), 4-hydrotamoxifen (4-HT) or liposome-complexed DNA constructs coding for antiangiogenic/anti-invasion proteins (angiostatin, TIMP-2, IFN-a 1 , sFLT-1) were individually administered to MMTVneu transgenic mice. Significant inhibition of primary tumor growth was obtained with TAM (40% inhibition, P¼0.049), angiostatin (85% inhibition, P¼0.001) and TIMP-2 (60% inhibition, P¼0.015). No lung metastasis was observed in any of these treated mice at 5 months, compared with a rate of 70% in control groups. These observations were the basis for designing a combined treatment with all these compounds. The association of angiostatin, TIMP-2 and TAM was greatly effective at the primary tumor level (90% inhibition, P¼0.01). Moreover, all the mice treated with this association were metastasis free at a time point (6 months) in which seven out of nine control mice were either dead from disseminated cancer or showed lung metastasis. This combined therapy could become an important component of anticancer therapy in humans.

Oncogene-bearing transgenic mice develop various kinds carrying an antisense anti-neu construct. This inhibitory of tumors depending on both the regulatory sequences and effect is specific, as it is related to the expression of the the specific oncogene used. These mice not only help to antisense transgene (determined by RT-PCR), and to a clarify the pathogenetic pathways leading to tumor formareduction in neu mRNA and protein, as determined by tion, but can also be useful as models to test novel theraNorthern and Western blot analyses. Moreover, inoculation peutic strategies, including gene therapy. We have preof cells carrying the antisense or the control vector in nude viously reported the establishment of an MMTV-neu (ErbBmice demonstrated that the morphological and biochemical 2) transgenic mouse lineage, in which 100% of females effects elicited by the antisense construct resulted in a sigdevelop breast tumors with many features similar to their nificantly slower rate of in vivo growth of tumor xenografts. human counterparts; these tumors are due to the overFinally, significant mammary tumor growth inhibition was expression of the activated rat neu oncogene in the mamobtained after liposome-mediated direct inoculation of the mary gland. From one such mouse we established a cell same antisense vector in tumors spontaneously arising in line of mammary adenocarcinoma named MG1361. We MMTV-neu mice. Taken together, these findings suggest that report here that the growth of this cell line can be inhibited targeting neu expression by an integrated large anti-neu antiin vitro and in vivo by transfection of a plasmid vector sense segment affects the in vivo growth of these tumors.

The Insulin Receptor (IR) is a potential oncogene for mammary epithelial cells since its content is increased in most human breast cancer specimens, and both ligand-dependent malignant transformation and ligand-dependent enhanced growth occurs in cultured breast cells overexpressing the IR. To better understand whether the IR plays a role in mammary carcinogenesis which is independent of other initiation factors, we measured IR content in transgenic mouse models of breast cancer induced by 3 known oncogenes (Wnt-1, Neu, and Ret). Insulin receptor content was measured by a specific radioimmunoassay. In normal mammary gland tissues IR content was 14.6 +/- 1.4 ng/mg of protein (mean +/- SEM, n = 6). In the 3 cancers IR content was elevated (Neu = 36.1 +/- 4.6, n = 8, p< 0.002; Wnt-1 = 38.3 +/- 2.6, n = 13, p < 0.001; and Ret = 53.6 +/- 7.1, n = 7, p < 0.001). These data indicate that IR overexpression, in addition to being a potential oncogene, is increased in mouse tumors initiated by other oncogenes, and therefore may also play a supportive role in the growth of breast cancers.