多品系多部位腹水癌(H22)淋巴转移动物模型的研究

目的：观察多品系小鼠多部位接种H22腹水癌能否引发淋巴结转移,以及它们的差异。方法：选择KM小鼠、BALB／c小鼠、C57BL／6小鼠,分别在腋下、股部内侧和脚垫接种H22腹水癌细胞,观察腋下和腹股沟淋巴结的变化。结果：腋下、股部内侧和脚垫接种组50d动物死亡率分别是：KM小鼠为100％、60％乘0％;BALB／c小鼠为80％、60％和0％;C57BL／6小鼠为100％、50％和0％。试验组动物的淋巴结重量普遍大于空白对照,尤其是KM小鼠和BALB／c小鼠脚垫接种组,病理检查显示,右腹股沟淋巴结可见大量癌细胞淋巴结的正常结构完全被破坏,甚至消失;C57BL,／6小鼠脚垫接种组淋巴结病理检查未发现癌细胞,淋巴结结构完整。结论：KM小鼠、BALB／c小鼠脚垫接种H22腹水癌细胞能够复制存活时间在50d以上,癌细胞发生淋巴结转移的动物模型。     

变性梯度凝胶电泳在实验小鼠细菌检测中的应用

目的 研究变性梯度凝胶电泳(denatured gradient gel electrophoresis, DGGE)在实验小鼠细菌检测中的应用。方法 根据16S rDNA V3区引物,PCR扩增3种实验小鼠(KM小鼠、NIH小鼠和BALB/c小鼠)呼吸道和盲肠段的细菌基因组DNA;扩增产物运用DGGE进行电泳检测,并分析条带数量间差异的统计学意义。结果 KM小鼠盲肠段条带12~18条,呼吸道条带5~10条;NIH小鼠盲肠段条带15~20条,呼吸道条带4~10条;BALB/c小鼠盲肠段条带10~15条,呼吸道条带0~7条。统计分析结果显示,KM小鼠和NIH小鼠在盲肠和呼吸道电泳条带数量上的差异无统计学意义( P >0.05);BALB/c小鼠与KM小鼠、NIH小鼠间的差异均有统计学意义( P <0.05)。结论 DGGE 在实验小鼠盲肠和呼吸道细菌检测中能较好地反映菌群的物种多样性。     

两品系小鼠食物过敏模型的比较

目的比较两种不同品系小鼠食物过敏模型的敏感性和肠道菌群变化的差异,旨在为食物过敏模型的建立提供依据。方法分别对30只4~5周龄BALB/c和KM雌鼠用卵清蛋白(ovalbumin,OVA)致敏建立食物过敏模型,ELISA法检测小鼠血清OVA特异性IgE水平;HE染色观察空肠组织形态;采用DGGE技术检测粪便菌群的变化。结果 (1)30只致敏的BALB/c小鼠中有27只血清OVA特异性IgE水平明显升高(P0.001),而30只致敏的KM小鼠中有21只,且BALB/c小鼠空肠绒毛炎症细胞浸润、上皮脱落及坏死比KM小鼠明显;(2)食物过敏造模后,BALB/c小鼠肠道菌群的改变明显(P0.001),而KM小鼠中仅有均匀度改变显著(P0.05);(3)BALB/c小鼠和KM小鼠对照组肠道菌群的丰富度、Shannon指数及均匀度都有差异。结论 BALB/c小鼠对OVA的敏感性高于KM小鼠,不同品系小鼠肠道菌群结构不同,OVA处理后,BALB/c小鼠菌群的改变比KM小鼠更明显。     

小鼠肺腺癌模型的建立及肿瘤病理分析

目的用乙基亚硝脲（ENU）在BABL/c小鼠建立肺腺癌模型并对ENU所诱发的肺腺癌进行病理观察。方法妊娠17d的SPF级母鼠腹腔接受ENU或缓冲液注射,在子代鼠的鼠龄满32周时收获其全肺标本,对肺组织进行常规石蜡半连续切片,HE染色,镜下观察肿瘤病理。结果 ENU经胎盘一次性诱发子代鼠多发性肺肿瘤形成,病理显示这些肿瘤为处于不同发展阶段的腺瘤和腺癌,腺癌的类型有细支气管肺泡癌样腺癌（雌性：5/6,雄性：4/6）和分化不等的腺癌（雌性：4/6,雄性：5/6）,诱癌频率在雌、雄性小鼠均为5/6,癌变频率在雌性16/43,雄性12/31。结论成功建立了小鼠肺腺癌模型,肿瘤病理的多样性提示癌变机制在分子水平的复杂性。     

艾氏腹水癌克隆细胞株E2G8建立小鼠动物模型生物学特性研究

目的　比较不同品种 (系 )小鼠接种E2G8细胞株的某些生物学特性。方法　将E2G8细胞稀释成不同浓度 ,接种KM、DBA 2、6 15、C57BL 6及BALB c小鼠皮下或腹腔 ,观察小鼠出现可见肿块 腹水时间及小鼠生存时间 ;测量肿块直径 (cm)及瘤重 ;荷瘤小鼠腹腔注射环磷酰胺 (CTX) ,观察E2G8细胞肿瘤模型对CTX治疗的敏感性。结果　E2G8接种BALB c、6 15小鼠皮下毒性最大 ,DBA 2、C57BL 6小鼠次之 ,接种KM小鼠皮下毒性最小 ;接种DBA 2、C57BL 6、6 15小鼠腹腔毒性较大 ,致死性高 ,对BALB c和KM小鼠的致死性弱 ;E2G8瘤细胞在KM和 4种纯系小鼠皮下均能很好生长肿瘤 ,其中在KM小鼠皮下肿瘤生长最大 ,在BALB c小鼠皮下肿瘤最小 ;KM、C57BL 6、6 15、DBA 2、BALB c小鼠皮下接种瘤细胞第 12天瘤重分别为 (2 4 0± 1 30 ) ,(0 90± 0 4 8) ,(1 2 0± 0 38) ,(1 10± 0 2 9) ,(0 80± 0 30 )g ;E2G8细胞接种 5个不同品种 (系 )小鼠制备的肿瘤模型 ,用CTX治疗 ,抑瘤率由高到低依次为6 6 7% (6 15 ) ,5 5 0 % (BALB c) ,5 3 3% (C57BL 6 ) ,5 0 % (KM) ,36 4 (DBA 2 ) ,其中 6 15小鼠建立的模型对CTX的治疗最敏感。结论　E2G8细胞株接种 5个不同品种 (系 )小鼠 ,所测生物学特性不完全相同 ,但是从腹腔 皮下接种、CTX     

SPF级小鼠超数排卵效果

目的探求小鼠周龄、品系和季节对SPF级小鼠超数排卵效果的影响。方法在春、夏、冬季对4～10周龄的SPF级BALB／c小鼠和KM小鼠进行超数排卵处理,通过见栓率、产胚率、3．5d胚胎获得量及胚胎类型等方面进行统计学分析。结果小鼠周龄、品系和季节对SPF级BALB／c小鼠和KM小鼠见栓率没有影响;对产胚率、3．5d胚胎获得量和胚胎类型等方面有一定影响。结论SPF级BALB／c小鼠宜用9、10周龄的进行超数排卵处理以供应3．5d胚胎为需要所用,而SPF级KM小鼠使用4～10周龄的都可以;另外,SPF级小鼠超数排卵处理仍宜在春、夏季进行,可以保证获得最佳的效果。     

SPF级KM小鼠与BALB/c小鼠肠道总菌多样性的比较

目的分析SPF级封闭群KM小鼠及近交系BALB/c小鼠的肠道菌群总菌多样性,比较两个不同遗传背景肠道总菌的丰富度、Shannon-Wiener指数和均匀度。方法收集SPF级KM小鼠和BALB/c小鼠新鲜粪便,提取粪便总菌DNA,用基于细菌16S rDNA序列的变性梯度凝胶电泳(PCR-DGGE)分析粪便总菌多样性。结果 SPF级KM小鼠及BALB/c小鼠粪便总菌多样性差异无统计学意义(P0.05),品系内不同性别之间粪便总菌多样性差异亦无统计学意义(P0.05)。结论选择SPF级小鼠进行微生态学相关研究时,封闭群KM小鼠及近交系BALB/c小鼠均可作为选择对象,同时可忽略菌群的性别差异。     

不同方式构建A20鼠B细胞淋巴瘤动物模型

目的探索多种方式构建A20鼠B细胞淋巴瘤动物模型及不同方式造模成瘤的特征。方法鼠源性B细胞淋巴瘤细胞株A20经皮下、尾静脉、脾脏和腹腔接种于同源BALB/c小鼠或先接种裸鼠成瘤后组织块移植BALB/c小鼠,观察动物成瘤时间、成瘤率、成瘤部位;取肿瘤组织和动物脏器行石蜡包埋、病理切片、HE染色观察其组织学特点。结果BALB/c鼠皮下注2×10^6组、2×10^7组和裸鼠瘤组织移植BALB/c小鼠组成瘤率皆为100%,成瘤时间分别为（15.29±3.2）d（、7.0±0.82）d和（6.29±0.49）d。BALB/c小鼠尾静脉注射2×106组、2×107组、脾脏注射组、腹腔注射组成瘤率分别为71.4%、100%、71.4%、14.3%,成瘤时间分别为（76.8±12.0）d、（26.1±7.99）d、（32.6±5.99）d和27 d。尾静脉成瘤部位播及肝脏、脾脏、胰腺、肾脏、食道、胃、肠、肠系膜、脑、淋巴结、骨、子宫、肌肉等多脏器和组织。BALB/c鼠A20成瘤组织学类似人弥漫大B细胞淋巴瘤。结论成功构建A20皮下移植瘤模型、血行播散性模型,为利用有免疫功能动物进行B淋巴瘤相关研究提供了实验平台。     

BALB/C等乳鼠流行性腹泻(EDIM)的初步研究

本文研究了1986—1989年间BALB/C小鼠发生的两次散发流行和一次有BALB/C及NIH鼠群暴发流行的EDIM(Epizootic diarrhea of infant mice)。经过电镜、PAGE、实验感染、“ABC”组化抗原定位等证实病原为MRV,而且病毒大小、RNA电泳型与国外报道的MRV有差异。观察了本繁育场内7个品系小鼠,均查出有MRV抗体,但发病只见于BALB/C和NIH鼠群。不同鼠龄和胎次发病率亦有差异。还观察了病变组织的病理组织学,扫描电镜和超微结构的改变。     

尾静脉注射EMT-6细胞建立转移性肺癌模型的研究

目的通过尾静脉注射EMT-6细胞建立一种临床特征明显、稳定可重复的转移性肺癌BALB/c小鼠模型。方法取对数生长期的EMT-6细胞,用0.25%胰蛋白酶消化,再用磷酸盐缓冲溶液调整细胞浓度至1×10~6个/m L(高)、5×10~5个/m L(中)和1×10~5个/m L(低)3个浓度,尾静脉注射0.2 m L EMT-6细胞悬液,对不同浓度模型的临床症状、成瘤时间、生存期、成瘤程度和病理学特征等生物学特征和评价指标进行研究,筛选出效果好的模型,并对该模型的重复性和稳定性通过3次重复实验加以确定,成功建立EMT-6细胞转移性肺癌BALB/c小鼠模型。结果 EMT-6细胞注射后解剖小鼠发现,高浓度组小鼠7 d肺表面开始出现肿瘤灶,18 d内所有小鼠死亡;中浓度组小鼠7 d肺表面开始出现肿瘤灶,28 d内所有小鼠死亡;低浓度组小鼠14 d肺表面开始出现肿瘤灶,21 d所有小鼠肺表面均有肿瘤灶,荷瘤小鼠24 d开始出现死亡,35 d肿瘤灶数量达到顶峰(平均12~15个/只)且肿瘤体积变大,42 d内全部死亡。结论相比之下,低浓度模型成瘤时间到小鼠全部死亡时间,为实验研究留有4周的处理、观察、评价的窗口期,其病理组织学符合肺癌的组织学特征,临床症状指标明显,模型重复性和稳定性好,是适合转移性肺癌研究的理想模型。     

Expression of the C-C chemokine receptor 7 mediates metastasis of breast cancer to the lymph nodes in mice

C-C chemokine receptor 7 (CCR7) controls lymphocyte migration to secondary lymphoid organs. Although CCR7 has been implicated in targeting the metastasis of cancers to lymph nodes, the role of CCR7 in the metastasis of breast cancer, along with the molecular mechanisms that are controlled by CCR7 that target breast cancer metastasis to the lymph nodes, has yet to be defined. To explore the cellular and molecular mechanisms of breast cancer cell migration to the lymph nodes, we used the mouse MMTV-PyVmT mammary tumor cells (PyVmT) transfected with CCR7 and the human CCR7-expressing MCF10A and MCF7 mammary cell lines. We found that the CCR7 ligands CCL19 and CCL21, controlled cell migration using the β(1)-integrin heterodimeric adhesion molecules. To define a physiological significance for CCR7 regulation of migration, we used the FVB syngeneic mouse model of metastatic breast cancer. When CCR7-negative PyVmT cells transfected with control vector were orthotopically transferred to the mammary fat pad of FVB mice, tumors metastasized to the lungs (10/10 mice) but not to the lymph nodes (0/10). In contrast, CCR7-expressing PyVmT (CCR7-PyVmT) cells metastasized to the lymph nodes (6/10 mice) and had a reduced rate of metastasis to the lungs (4/10 mice). CCR7-PyVmT tumors grew significantly faster than PyVmT tumors, which mirrored the growth in vitro, of CCR7-PyVmT, MCF7, and MCF10A mammospheres. This model provides tools for studying lymph node metastasis, CCR7 regulation of tumor cell growth, and targeting of breast cancer cells to the lymph nodes.     

Inhibition of the development of metastases by dietary vitamin C:K3 combination

The tumor growth-inhibiting and chemo-potentiating effects of vitamin C and K(3)combinations have been demonstrated both in vitro and in vivo. The purpose of this study was to investigate the influence of orally administered vitamin C and K(3) on the metastasis of mouse liver tumor (T.L.T.) cells implanted in C3H mice. Adult male C3H mice were given water containing vitamin C and K3 (15 g/0.15 g dissolved in 1000 ml) beginning 2 weeks before tumor transplantation until the end of the experiment. T.L.T. cells (106) were implanted intramuscularly in the right thigh of mice. All mice were sacrificed 42 days after tumor transplantation. Primary tumor, lungs, lymph nodes and other organs or tissues suspected of harboring metastases were macroscopically examined. Samples of primary tumors, their local lymph nodes, lungs and main organs such as liver, kidneys, spleen were taken for histological examination. Forty-two percent of control mice exhibited lung metastases and 27% possessed metastases in local lymph nodes whereas 24% of vitamin-treated mice exhibited lung metastases and 10% possessed local lymph nodes metastases. The total number of lung metastases was 19 in control group and 10 in vitamin C and K(3)-treated mice. Histopathological examination of the metastatic tumors from the vitamin-treated mice revealed the presence of many tumor cells undergoing autoschizic cell death. These results demonstrate that oral vitamin C and K(3) significantly inhibited the metastases of T.L.T. tumors in C3H mice. At least a portion of this inhibition was due to tumor cell death by autoschizis.     

Behavior of metastatic and nonmetastatic breast tumors in old mice

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.     

Pulmonary metastases in guinea pigs as a consequence of dermal implantation of line-10 tumor cells

Summary Metastases to the lungs of guinea pigs occurred at high frequency as a consequence of intradermal implantation of tumor cells derived from the syngeneic hepatocellular carcinoma line-10. Surgery had a major influence on the proportion of guinea pigs found to have pulmonary metastases at necropsy. Without surgery all guinea pigs died with extensive lymph node metastases; macroscopic pulmonary metastases were present in a minority of the animals. Animals treated by excision of dermal tumors survived longer than untreated animals, and macroscopic pulmonary metastases were present in the majority of the animals. Animals treated by excision of dermal tumor and regional lymph nodes were rendered tumor-free. The data suggest that lymph node metastases were the most likely source of the tumor cells that spread to the lungs in animals from whom the dermal tumor transplant had been removed.     

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.     

Allelic imbalance in primary breast carcinomas and metastatic tumors of the axillary lymph nodes

Axillary lymph node status is the most important prognostic factor in predicting disease outcome in women with breast cancer. A number of chromosomal aberrations in primary breast tumors have been correlated with lymph node status and clinical outcome, but chromosomal changes particular to metastatic lymph node tumors have not been well studied. DNA samples isolated from laser-microdissected primary breast and metastatic axillary lymph node tumors from 25 women with invasive breast cancer were amplified using 52 microsatellite markers defining 26 chromosomal regions commonly deleted in breast cancer. Levels and patterns of allelic imbalance (AI) within and between breast and lymph node tumors were assessed to identify chromosomal alterations unique to primary or metastatic tumors and to examine the timing of metastatic potential. The overall frequency of AI in primary breast tumors (0.24) was significantly greater (P < 0.001) than that in lymph node tumors (0.10), and congruent AI events were observed for < 20% of informative markers. AI at chromosomes 11q23.3 and 17p13.3 occurred significantly more frequently (P < 0.05) in primary breast tumors alone; no chromosomal regions showed a significantly higher AI frequency in lymph nodes. Higher rates of AI in primary versus metastatic lymph node tumors suggest that acquisition of metastatic potential may be an early event in carcinogenesis, occurring before significant levels of AI accumulate in the primary tumor. In addition, patterns of AI were highly discordant between tumor types, suggesting that additional genetic alterations accumulated independently in the two cell populations.     

Further characterization of a clinically relevant model of melanoma metastasis and an effective vaccine

A major problem in evaluating the effectiveness of tumor cell vaccination and other biological therapies is the variability of experimental models. In this study we have further developed and characterized a model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination: stage I-growth of the primary (local) tumor, stage II-dissemination to regional lymph nodes, and stage III-metastasis to distant organs (lungs). C57BL/6 mice were challenged subcutaneously with B16 F10 murine melanoma cells in the midtail, and within 3 weeks 100% of the mice had local tumors growing in their tails. By 5–7 weeks after challenge, most of the mice had developed metastases to the inguinal lymph nodes and subsequently had metastatic colonies in the lungs and in the bone marrow. Preimmunization of mice with a formalinized extracellular antigen vaccine, derived from B16F10 melanoma cells, provided partial inhibition of the growth of the primary melanoma tumors, as well as reducing the number of metastases to the regional (inguinal) lymph nodes and lungs along with concomitantly increasing survival time. This model for melanoma metastasis provides a reasonable and reproducible test system for the study of anti-melanoma immunity and the different cellular and humoral mechanisms involved.This work was supported in part by National Institutes of Health grants R37 CA45148 and R30 CA13943     

Lower expression of CXCR4 in lymph node metastases than in primary breast cancers: potential regulation by ligand-dependent degradation and HIF-1alpha

Stromal-derived factor-1 (SDF-1) is a unique ligand of the CXC chemokine receptor 4 (CXCR4), which is critically involved in the metastasis of breast cancer. High levels of SDF-1 in the common destination organs of metastasis, such as the lymph nodes, lungs, liver, and bones, attract CXCR4-positive tumor cells. The interaction between SDF-1 and CXCR4 leads to the activation of specific signaling pathways, allowing for homing and metastatic progression. However, regulation of CXCR4 expression at the metastatic organ site is not well-documented. We detected the expression of CXCR4 and hypoxia inducible factor (HIF)-1alpha in breast tumor tissues by immunohistochemical staining and analyzed SDF-1 in primary tumors and lymph nodes using real-time RT-PCR. Compared to the corresponding metastasized tumors in the lymph nodes, primary invasive carcinomas showed more intense staining for CXCR4, particularly on the cellular membrane. Both primary tumors and lymph node metastases exhibited higher levels of CXCR4 expression compared to non-neoplastic breast tissues. Therefore, we hypothesized that the tumor environment in the lymph nodes may cause the reduction of CXCR4 levels in the metastatic tumor cells because of: (1) high SDF-1 levels and (2) lower levels of HIF-1alpha. Our in vitro data demonstrated that high levels of SDF-1 can induce the internalization and degradation of CXCR4 through the lysosome pathway. In addition, lower levels of HIF-1alpha in the lymph node metastases, probably induced by the less hypoxic environment, further lowered CXCR4 levels. These results indicate that ligand-dependent degradation and lower HIF-1alpha levels may be potential causes of lowered levels of CXCR4 in the lymph nodes compared to the primary tumors. Our study suggests that CXCR4 levels in tumor cells are regulated by its microenvironment. These findings may enhance our ability to understand the biological behavior of breast cancers.     

Calorie consumption level influences development of C3H/Ou breast adenocarcinoma with indifference to calorie source

To analyze simultaneously the influence attributable to calorie consumption level and percentage of dietary fat on the spontaneous development of mammary adenocarcinoma, virgin female C3H/Ou mice were separated into five dietary groups. Four groups of mice were fed purified diets either ad libitum (16-18 kcal/mouse/day) or restricted 40% in calorie consumption (10-11 kcal/mouse/day), and diets contained either 4.5%, 7.5%, 67%, or 68% calories from fat. Mice that consumed isocaloric diets developed breast malignancy at a comparable pace. Consuming a diet in which fats were present only at levels sufficient to satisfy the threshold requirement of essential fatty acids, 4.5-7.5% of the total calories, or alternatively where dietary fat represented greater than 67% of the total calories consumed, did not significantly alter the tendency for breast tumor development. The pace and frequency with which tumors occurred reflected the host's level of calorie consumption. Mice consuming a high caloric diet, low or high in fat, tended to have a shortened latency to breast tumor formation, an increased incidence of breast tumors, elevated serum prolactin levels, elevated levels of antibodies to mouse mammary tumor virus, and elevated circulating immune complex levels.     

Raman spectroscopy can differentiate malignant tumors from normal breast tissue and detect early neoplastic changes in a mouse model

Raman spectroscopy shows potential in differentiating tumors from normal tissue. We used Raman spectroscopy with near-infrared light excitation to study normal breast tissue and tumors from 11 mice injected with a cancer cell line. Spectra were collected from 17 tumors, 18 samples of adjacent breast tissue and lymph nodes, and 17 tissue samples from the contralateral breast and its adjacent lymph nodes. Discriminant function analysis was used for classification with principal component analysis scores as input data. Tissues were examined by light microscopy following formalin fixation and hematoxylin and eosin staining. Discriminant function analysis and histology agreed on the diagnosis of all contralateral normal, tumor, and mastitis samples, except one tumor which was found to be more similar to normal tissue. Normal tissue adjacent to each tumor was examined as a separate data group called tumor bed. Scattered morphologically suspicious atypical cells not definite for tumor were present in the tumor bed samples. Classification of tumor bed tissue showed that some tumor bed tissues are diagnostically different from normal, tumor, and mastitis tissue. This may reflect malignant molecular alterations prior to morphologic changes, as expected in preneoplastic processes. Raman spectroscopy not only distinguishes tumor from normal breast tissue, but also detects early neoplastic changes prior to definite morphologic alteration.