A stem cell strategy to induce tumor stasis and inhibit metastasis of breast canc

描述（由申请人提供）：乳腺癌细胞的恶性表型可以通过胚胎环境逆转为正常细胞表型，突出了治疗恶性乳腺癌而不是杀死癌细胞的可能的新型细胞转化策略。诱导多能干细胞（iPSC）技术可以将恶性癌细胞重编程为胚胎干细胞（ESC）样细胞，提供活的胚胎环境。然而，目前iPSC技术的低转化率限制了其在癌症研究和治疗中的广泛应用。我们开发了一种蛋白诱导的PSC（piPSC）技术，其使用Sox 2/Oct 4/Nanog（SON）蛋白以90 - 4%的转化率将异质性恶性乳腺癌细胞（malBCC）重编程为piPSC。将4 T1-piPSC植入小鼠乳腺中导致肿瘤停滞、转移抑制和分化成正常乳腺细胞。4 T1细胞与4 T1-piPSC的共培养表明旁观者效应，其使得少量4 T1-piPSC能够逆转大量周围4 T1细胞的恶性表型。在4 T1荷瘤小鼠中直接肿瘤内注射“SON”蛋白导致肿瘤停滞和转移抑制，表明原位细胞转化癌症疗法。QQ-SON蛋白的尾静脉注射将这些蛋白靶向到乳腺癌的肿瘤细胞的细胞核中。我们的中心假设是：“SON”蛋白的注射将malBCC原位转化为piPSC，其通过piPSC的旁观者效应将周围malBCC的恶性表型逆转为正常细胞”。我们将首先优化从乳腺上皮细胞和malBCC产生piPSC。将进行malBCC与piPSC的共培养以研究是否可以在体外降低共培养的malBCC的致癌特性（目的1）。为了研究体内的旁观者效应，我们将施用不同的 在不同的时间点将不同剂量的（少量）piPSC注入荷瘤小鼠的肿瘤（大量）中，以研究这些piPSC是否可以诱导肿瘤停滞并抑制体内转移（目的2）。为了模拟乳腺癌治疗的临床环境，我们将直接注射 在不同的时间点，通过尾静脉或通过肿瘤内将“SON”蛋白导入乳腺癌荷瘤小鼠中，以研究原位细胞转化是否可以诱导肿瘤停滞并抑制转移，从而延迟乳腺癌复发并延长生存期（目的3）。与目前基于分子的细胞杀伤疗法不同，我们将产生组织特异性 piPSC原位作为活胚胎环境以将乳腺癌细胞的恶性表型逆转为正常细胞。这将通过经由尾静脉和经由肿瘤内将SON蛋白注射到乳腺癌转移性小鼠模型中来实现。虽然该提议侧重于原理验证，但蛋白质诱导的原位细胞转化策略将容易地转化为人类临床应用。该提案的成功可能会为这种拟议的细胞转化乳腺癌疗法的未来人类临床试验奠定基础，通过显着延迟癌症复发，延长生存期和改善乳腺癌患者的预后。

DESCRIPTION (provided by applicant): Malignant phenotypes of breast cancer cells can be reversed into normal cell phenotypes by embryonic environments, highlighting a possible novel cell-converting strategy to treat malignant breast cancers instead of killing cancerous cells. The induced Pluripotent Stem Cell (iPSC) technique can reprogram malignant cancer cells into Embryonic Stem Cell (ESC)-like cells, providing living embryonic environments. However, the low conversion of the current iPSC technique limits its broad applications in cancer research and treatment. We developed a protein-induced PSC (piPSC) technique that reprogrammed heterogeneous malignant Breast Cancer Cells (malBCCs) into piPSCs using the Sox2/Oct4/Nanog (SON) proteins with 90¿4% conversion. Implantations of the 4T1-piPSCs into mouse mammary glands resulted in tumor stasis, metastasis inhibition and differentiation into normal mammary cells. Co-cultures of 4T1 cells with 4T1-piPSCs suggest a bystander effect that enables a small number of 4T1-piPSCs to reverse malignant phenotype of large numbers of surrounding 4T1 cells. Direct intra-tumor injections of the "SON" proteins in the 4T1 tumor bearing mice resulted in tumor stasis and metastasis inhibition, suggesting an in situ cell-converting cancer therapy. Tail vein injections of QQ-SON proteins targeted these proteins into the nuclei of tumor cells of breast cancer. Our central hypothesis is: "Injections of the "SON" proteins convert malBCCs into piPSCs in situ that reverse malignant phenotype of the surrounding malBCCs into normal cells via a bystander effect of piPSCs". We will first optimize piPSC generation from mammary epithelial cells and malBCCs. Co-cultures of malBCCs with piPSCs will be performed to study if oncogenic properties of the co-cultured malBCCs can be reduced in vitro (Aim 1). To investigate the bystander effect in vivo, we will administer different doses of (small numbers) piPSCs into the tumor (large numbers) of the tumor-bearing mice at different time points to study if these piPSCs can induce tumor stasis and inhibit metastasis in vivo (Aim 2). To mimic the clinical setting of breast cancer treatment, we will directly inject the "SON" proteins either via tail vein or via intra-tumor into the breast cancer-bearing mice at different time points to investigate if the in situ cell conversion can induce tumor stasis and inhibit metastasis, therefore delaying breast cancer recurrence and prolonging survival (Aim 3). Different from the current molecular-based cell-killing therapies, we will generate tissue-specific piPSCs in situ as living embryonic environments to reverse malignant phenotype of breast cancer cells into normal cells. This will be achieved by injections of the SON proteins via tail vein and via intra-tumor into breast cancer metastatic mouse models. Although this proposal focuses on a proof-of-principle verification, a protein-induced in situ cell-conversion strategy wil be easily translatable to human clinical applications. The success of this proposal may set a stage for future human clinical trials of this proposed cell converting breast cancer therapy by significantly delaying cancer recurrence, prolonging survival and improving outcomes of breast cancer patients.