Tumor Stroma Interactions: Wound Promoted Tumor Growth

伤口促进肿瘤发生和肿瘤进展的机制尚不完全清楚。特别是，急性伤口的影响，如可能发生在活组织检查或探查手术对现有肿瘤的影响尚未得到解决。也许最大的风险是肿瘤切除后留下的未被怀疑的肿瘤微沉积物。迄今为止，动物模型已经描述了先前存在的伤口对肿瘤细胞的影响，或者伤害对初始宿主的影响。例如，与未受伤的组织相比，将黑色素瘤或纤维肉瘤细胞注射到伤口中，肿瘤发病率和肿瘤体积更高，这表明先前存在的伤口微环境有助于肿瘤细胞接种形成肿瘤。同样，伤口液和黑色素瘤细胞共同注射导致肿瘤体积增加。研究还表明，在劳斯肉瘤病毒感染的鸡和v-ras转基因小鼠中，全层经皮损伤是肿瘤表达和生长的充分条件。这表明，在已经由病毒感染或癌基因表达引发的宿主中，损伤可以促进肿瘤的发生。在劳斯肉瘤鸡模型中，tgf - β，一种多效细胞因子，被认为是伤口引发肿瘤发生的分子机制。这些报道表明，伤口微环境与肿瘤微环境之间存在很强的相似性和相互作用，这种相互作用可以加速肿瘤的发生和进展，对宿主不利。临床上，外科手术通常在已有肿瘤附近进行，作为肿瘤治疗的必要组成部分。虽然这些手术试图为了患者的利益而根除肿瘤，但局部肿瘤复发和肿瘤细胞沿伤口或针道植入已经被描述过。虽然这通常归因于肿瘤的机械扩散，但局部伤口环境本身也可能以消极的方式影响任何残留的肿瘤细胞。了解伤口-肿瘤相互作用的机制将有助于确定治疗靶点，以防止此类手术对肿瘤患者的负面影响。为了了解免疫系统在伤口促进肿瘤生长中的作用，我们开始研究伤口如何影响免疫功能低下动物的肿瘤生长。在胸腺BALB/c nu/nu小鼠实验中，我们发现伤口对肿瘤生长没有明显的促进作用，说明伤口促进肿瘤生长是由t细胞介导的。我们进一步证明，在将BALB/c小鼠产生的伤口液注射到动物体内之前，或在肿瘤部位附近注射伤口液，伤口液不仅可以提高体外增殖率，还可以加速体内肿瘤生长；BALB/c nu/nu动物创面液对肿瘤细胞增殖和肿瘤生长无显著影响。这些数据表明伤口促进肿瘤生长是由t淋巴细胞分泌的一种可溶性因子介导的。我们目前的目标是澄清不同t细胞亚群在我们的模型中的作用，并与Lalage Wakefield合作开始了CD8消耗实验。如果适用，我们将使用类似的方法通过耗尽来研究其他t细胞亚群，如CD4细胞。同时，我们将在受伤后的不同时间点收集肿瘤组织，并使用免疫组织化学和FACS分析t细胞亚群流入肿瘤/伤口微环境。由于我们已经证明了伤口液对体内肿瘤细胞增殖和体外肿瘤生长的影响，我们将使用抗体微阵列（Raybiotech）分析BALB/c、BALB/c nu/nu小鼠、小鼠血浆和小鼠血清产生的伤口液的细胞因子表达模式；蛋白质表达模式将通过2d电泳和随后的质谱分析，如果适用，将使用蛋白质微阵列。基于这些方法和现有文献，我们将建立一个候选细胞因子或蛋白的候选名单，介导伤口促进肿瘤生长。或者，我们将开始分离伤口液，并研究这些部分对肿瘤细胞和基质细胞（如成纤维细胞或内皮细胞）的影响，以确定新的效应分子。此外，tgf - β与劳斯肉瘤病毒感染鸡的伤口引发的肿瘤发生有关。tgf - β在肿瘤发生和转移中具有复杂的作用，其对不同细胞类型的影响取决于细胞类型和信号环境。例如，tgf - β可以刺激基质细胞和血管生成的基质分泌，并调节免疫功能，所有这些都在肿瘤发生和伤口愈合过程中发生改变。使用Smad3敲除模型，我们可以证明在4T1模型中，基质tgf - β信号缺陷产生更小的肿瘤，并且Smad3敲除动物的肿瘤具有更少的CD31阳性血管。我们将Smad3基因敲除培养到BALB/c背景下，分析基质tgf - β信号缺陷对伤口促进肿瘤生长的影响。使用这个模型，我们能够确定几个候选蛋白参与伤口促进肿瘤生长。特别是，我们证明了SDF-1预处理细胞会增加肿瘤生长，而抑制SDF-1和/或其受体CXCR4会降低肿瘤生长。研究还表明，体内抑制SDF-1/CXCR4信号传导可降低伤口对肿瘤的影响。我们现在计划在伤口/肿瘤中确定SDF-1的来源，研究SDF-1在伤口/肿瘤促进生长中的作用，并确定SDF-1的靶细胞。此外，我们将通过识别伤口中的t淋巴细胞亚群和识别哪些t淋巴细胞亚群介导伤口促进肿瘤生长来追踪t淋巴细胞在伤口促进肿瘤生长中的作用。

The mechanisms of wound promoted tumorigenesis and tumor progression are not fully understood. In particular, the impact of an acute wound such as might occur during biopsy or explorative surgery on an existing tumor has not been addressed. Perhaps the greatest risk is the presence of unsuspected micro deposits of tumor left behind after tumor resection. Animal models to date have described the influence of preexisting wounds on tumor cells, or the influence of wounding on initiated hosts. For example, tumor incidence and tumor volume are higher if melanoma or fibrosarcoma cells are injected into a wound as compared to unwounded tissue, indicating that a preexisting wound microenvironment facilitates the establishment of tumors from a tumor cell inoculate. Likewise, the coinjection of wound fluid and melanoma cells resulted in increased tumor volumes. It has also been shown that full thickness transcutaneous wounding is a sufficient event for tumor expression and growth in both Rous-sarcoma virus infected chickens and v-ras transgenic mice. This demonstrates that wounding can promote tumorigenesis in a host that is already initiated by viral infection or by oncogene expression. In the Rous sarcoma chicken model TGF-beta, a pleiotropic cytokine, has been implicated as a molecular mechanism of wound initiated tumorigenesis. These reports suggest a strong similarity as well as interaction between the wound microenvironment and the tumor microenvironment, and that this interaction can accelerate tumorigenesis and tumor progression in an unfavorable way for the host. Clinically, surgical procedures are typically performed in the proximity of a pre-existing tumor as a necessary component of tumor treatment. While these procedures attempt to eradicate the tumor for the benefit of the patient, local tumor recurrence and implantation of tumor cells along the wound or the needle tract have been described. Although this is often attributed to mechanical tumor spread, the local wound environment itself may similarly influence any residual tumor cells in a negative way. Understanding the mechanisms involved in wound-tumor interactions will help to identify therapeutic targets to prevent a negative impact of such procedures on tumor patients. In order to understand the role of the immune system in wound promoted tumor growth we have begun to study how wounding influences tumor growth in immunocompromised animals. Using athymic BALB/c nu/nu mice we found that wounding does not significantly accelerate tumor growth, indicating that wound promoted tumor growth is mediated by T-cells. We furthermore could show that wound fluid not only increased proliferation rates in vitro, but also accelerated tumor growth in vivo when 4T1 cells were treated with wound fluid generated from BALB/c mice before injection into animals, or when wound fluid was injected in the proximity of the tumor site; wound fluid generated in BALB/c nu/nu animals had no significant effect on tumor cell proliferation or tumor growth. These data indicate that wound promoted tumor growth is relayed by a soluble factor secreted by T-lymphocytes. We currently aim to clarify the role of different T-cell subsets in our model, and started CD8 depletion experiments in collaboration with Lalage Wakefield. If applicable, we will use a similar approach to investigate other T-cell subsets such as CD4 cells by depletion. In parallel, we will harvest tumor tissue at different time points after wounding and analyze the influx of T-cell subsets into the tumor / wound microenvironment using immunohisochemistry and FACS analysis. Since we already demonstrated that wound fluid effects proliferation of tumor cells in vivo and tumor growth in vitro, we will analyze the cytokine expression pattern of wound fluid generated from BALB/c, BALB/c nu/nu mice, mouse plasma and mouse serum using antibody microarrays (Raybiotech); protein expression patterns will be analyzed by 2D-electrophoresis and subsequent mass-spectometry, and protein microarrays will be employed if applicable. Based on these approaches and the current literature, we will establish a shortlist of candidate cytokines or proteins that mediate wound promoted tumor growth. Alternatively, we will start fractionating wound fluid and investigate the effect of these fractions on tumor cells and stromal cells such as fibroblasts or endothelial cells in order to identify new effector molecules. Furthermore TGF-beta has been implicated in wound triggered tumorigenesis in Rous sarcoma virus infected chickens. TGF-beta's has a complex role in tumorigenesis and metastases, and its influence on different cells types depends on the cell type and the signaling context. For example, TGF-beta can stimulate matrix secretion by stromal cells and angiogenesis, and modulates immune function, all of which are altered during tumorigenesis as well as during wound healing. Using a Smad3 knockout model we could show that defect stromal TGF-beta signaling yields smaller tumors in the 4T1 model, and that tumors from Smad3 knockout animals have less CD31 positive vessels. We bred the Smad3 knockout into the BALB/c background to analyze the effect of defective stromal TGF-beta signaling on wound promoted tumor growth. Using this model, we were able to identify several candidate proteins involved in wound promoted tumor growth. In particular, we demonstrated that pretreatment of cells with SDF-1 increased tumor growth while the inhibition of SDF-1 and/or its receptor CXCR4 decreased tumor growth. It was also demonstrated that the inhibition of SDF-1/CXCR4 signaling in vivo reduces the effect of wounds on tumors. We now plan to identify the origin of SDF-1 in wounds/tumors, investigate the role of SDF-1 in wounded promoted tumor growth and to identify the target cell of SDF-1. In addition, we will follow up on the role of T-lymphocytes in wound promoted tumor growth by identifying subsets of T-lymphocytes in wounds and identifying which subset of T-lymphocytes relays wound promoted tumor growth.