BioVector® M207 (Mel207) Human Malignant Melanoma Cell Line / M207 (Mel207) 人恶性黑色素瘤细胞系

一 产品基本信息与遗传学背景

• 细胞名称：M207（常写作 Mel207），人恶性黑色素瘤细胞。

• 物种来源：人源（Homo sapiens）。该细胞系是国际黑色素瘤转化医学研究中的重要基础模型，也是美国 MD 安德森癌症中心细胞系项目（MD Anderson Cell Lines Project）的代表性肿瘤株系之一（Cellosaurus 登录号：CVCL_D747）。

• 遗传学与分子突变特征（核心科研背景）：

  • NRAS 突变（关键特征）：M207 细胞携带经典的 NRAS 基因激活热点突变（p.Gln61Leu / c.182A>T）。大约有 15%–20% 的皮肤黑色素瘤由 NRAS 突变驱动。该突变导致下游的 MAPK 信号通路（RAS-RAF-MEK-ERK）和 PI3K/Akt/mTOR 通路发生强烈的、非配体依赖性的结构性持续激活。

  • EGFR 突变/变异：携带表皮生长因子受体 EGFR 基因激酶区缺失突变（p.Leu747_Pro753delinsSer），这在黑色素瘤群体中较为独特，常被用于探究黑色素瘤中受体酪氨酸激酶（RTK）旁路对常规疗法的耐药贡献。

  • PTEN 杂合缺失：具有抑癌基因 PTEN 的杂合性缺失（Heterozygous Deletion）。PTEN 的部分失活进一步解除了对 PI3K 通路的负反馈抑制，协同增强了该细胞的增殖与抗凋亡能力。

  • BRAF 状态：属于 BRAF 野生型（BRAF-wildtype）。该特征使其与临床最常见的 $BRAF^{V600E}$ 突变细胞系（如 A375、M14）形成鲜明的对照。

• 生长动力学与特性：贴壁生长（Adherent），细胞形态多呈现典型的多角形、梭形或不规则的上皮样（Epithelial-like）交织排布。其倍增时间（Doubling time）约为 25.2 小时，属于分裂扩增较为旺盛的侵袭性肿瘤模型。

• 生物安全级别：1-2 级（BSL-1/2）。操作应在标准的二级生物安全柜内严格按照无菌细胞房规范进行。

(注：在早期部分眼科文献或旧数据库分类中，该编号偶尔与眼葡萄膜黑色素瘤株系 Mel270 发生拼写混淆，但根据国际细胞鉴定委员会（ICLAC）与 Cellosaurus 权威认定，M207 是一株明确携带 NRAS 突变的恶性黑色素瘤细胞系。)

二 核心科研价值与转化医学应用

M207 细胞在攻克黑色素瘤药物耐药性及联合靶向方案设计中扮演了关键角色：

1. NRAS 突变型黑色素瘤靶向治疗与联合用药筛选：

   临床上针对 $BRAF^{V600E}$ 突变的黑色素瘤已有高效的维罗非尼（Vemurafenib/PLX4032）等靶向药，但这些药物对 NRAS 突变的 M207 细胞天然耐药。因此，M207 被广泛用作“反向对照株”，用于开发专门针对 NRAS 突变群体的广谱 RAF 抑制剂（Pan-RAF Inhibitor）联合 MEK 抑制剂（如 TAK733、Trametinib）的协同致死毒杀矩阵，或评价全新的 ERK 激酶小分子阻断剂。

2. 癌症反相蛋白质阵列（RPPA）与功能蛋白质组学：

   作为 MD 安德森癌症中心黑色素瘤项目的核心分析株，M207 拥有极度完善的反相蛋白质微阵列（RPPA）表达谱数据。科研人员可直接调取其在基础状态下或药物刺激后，数百种核心激酶（如 p-ERK、p-Akt、p-S6）的磷酸化动力学图谱，极其适合结合生物信息学研究肿瘤网络重构。

3. 肿瘤侵袭与微环境黏附研究：

   由于其携带 PTEN 缺陷与 NRAS 强激活，该细胞表现出极高的体外迁移、侵袭与胶原基质穿透能力，常用于构建划痕愈合模型、Transwell 侵袭实验，以及探究黑色素瘤细胞与血管内皮细胞、成纤维细胞之间的旁分泌交互。

三 实验室细胞复苏、扩增传代与冷冻保存标准步骤

1. 完全培养基配置

M207 细胞对营养基质适应性好，推荐配置如下：

• 基础培养基：RPMI 1640 培养基（推荐）（含 2.0 mM L-Glutamine 谷氨酰胺）。

• 完全添加剂成分：

  • 10% 优质灭活胎牛血清（FBS）。

  • 1% 灭菌双抗（Penicillin-Streptomycin, 100 U/mL）。

• 培养环境：37 摄氏度、5% $CO_2$、恒湿有氧培养箱。

2. 细胞复苏步骤

1. 将配置好的完全培养基在 37 摄氏度水浴中提前预热，准备 15 mL 离心管。

2. 从液氮罐或 -80 摄氏度冰箱中迅速取出 M207 冻存管，立刻投入 37 摄氏度恒温水浴箱中，轻柔规律地水平晃动。

3. 在 1 分钟之内令管内冻存块急速融化（待内部剩下最后一粒细小冰芯时即可捞出）。用 75% 酒精擦拭管外壁消毒后移入安全柜。

4. 用移液枪将融化的细胞悬液吸出，缓慢滴入含有 5 mL 预热完全培养基的离心管中，轻柔吹打混匀，防止瞬时渗透压过高损伤细胞膜。

5. 以 200 - 300 x g（约 1000 rpm）离心 3 - 4 分钟，小心吸除含 DMSO 的上清液。

6. 加入 5 mL 新鲜完全培养基重悬沉淀，接种至 T25 培养瓶中，置于 37 摄氏度孵箱中，次日观察贴壁展平率。

3. 细胞传代与消化方法

• 传代时机：由于 M207 增殖较为迅速（倍增约 25 小时），当细胞融合度达到 70% - 80% 时必须及时传代。若贴壁过密，NRAS 持续激活导致的局部代谢产物积聚会引发非特异性接触抑制或自发性漂浮。

• 传代比例：通常按照 1:3 至 1:5 的比例进行分瓶传代，每 2 - 3 天传代一次。

• 操作步骤：

  1. 吸干瓶内旧培养基，用无菌无钙镁 PBS 轻轻洗涤细胞表面 1 - 2 次，彻底洗去血清。

  2. 加入适量 0.25% Trypsin-EDTA 消化液（T25 瓶常规加入 1 mL），使其均匀浸润细胞层。

  3. 放入 37 摄氏度孵箱中消化 1 至 3 分钟。在倒置显微镜下连续观察，当大部分多角形细胞回缩变圆、边界透亮且轻击瓶壁细胞开始大量向下滑落时，立即加入 2 倍体积的含血清完全培养基终止消化。

  4. 用移液枪轻柔平稳地吹打瓶壁，将细胞吹散成单细胞悬液。收集至离心管中，300 x g 离心 3 分钟，弃上清。

  5. 加入新鲜完全培养基，吹匀后按比例接种到新的培养容器中。

4. 细胞冷冻保存

• 冻存液配方：90% 完全培养基（或纯高品质 FBS） + 10% 细胞级 DMSO。

• 冻存操作：冷冻前确保细胞处于对数生长活跃期（融合度约 75% 且活力达 90% 以上）。离心收集细胞，用配置好的冻存液重悬，调整最终细胞密度至 $1.5 \times 10^6$ 至 $3 \times 10^6\text{ cells/vial}$。装入冻存管后立即放入程序降温盒（如 Mr. Frosty 梯度降温盒，维持 -1 摄氏度/分钟的慢冻速度），置于 -80 摄氏度过夜。次日必须迅速转移至 -196 摄氏度液氮罐中长期冷冻保存，以防止细胞活力衰退。

Part 2 English Section

I General Information and Genetic Architecture

• Cell Line Designation: M207 (frequently indexed as Mel207), Human Malignant Melanoma Cell Line.

• Species Origin: Homo sapiens (Human).This lineage serves as a vital model for translational melanoma oncology and represents a core matrix within the MD Anderson Cancer Center Cell Lines Project (Cellosaurus Accession: CVCL_D747).

• Genomic and Mutational Profiles (Critical Research Framework):

  • NRAS Hyper-activation (Canonical Driver): M207 harbors a classical, canonical activating hotspot mutation in NRAS (p.Gln61Leu / c.182A>T). Approximately 15%–20% of cutaneous melanomas are driven by oncogenic NRAS variants. This driver leads to robust, ligand-independent constitutive hyper-activation of downstream MAPK cascades (RAS-RAF-MEK-ERK) and PI3K/Akt/mTOR pathways.

  • EGFR Variant Co-occurrence: Features an epidermal growth factor receptor EGFR kinase domain deletion mutation (p.Leu747_Pro753delinsSer). This configuration is rare in cutaneous melanoma, making M207 an excellent model to explore how receptor tyrosine kinase (RTK) feedback bypass loops contribute to therapeutic resistance.

  • PTEN Heterozygous Loss: Characterized by a heterozygous deletion of the tumor suppressor PTEN. Partial PTEN inactivation removes key negative feedback controls over the PI3K network, synergizing with the NRAS mutation to accelerate cell proliferation and evade apoptosis.

  • BRAF Wildtype Matrix: Characterized as BRAF-wildtype, making it an essential negative control line when compared against clinical $BRAF^{V600E}$ models (such as A375 or M14).

• Growth Kinetics & Morphology: Adherent monolayer. Displays distinct polygonal, spindle-shaped, or irregular epithelial-like interlaced cellular morphologies. It exhibits highly active proliferation kinetics with a documented doubling time of approximately 25.2 hours.

• Biosafety Matrix: Classified under Biosafety Level 1 or 2 (BSL-1/2). Standard handling protocols should proceed within certified Class II Biosafety Cabinets under sterile clinical culture conditions.

(Note: Historical literature occasionally notes minor typographical confusion between M207 and the uveal melanoma cell line Mel270. However, current authenticated data from ICLAC and Cellosaurus clarify that M207 is a distinct cutaneous malignant melanoma cell line harboring an NRAS mutation.)

II Strategic Research Value and Translational Fields

The M207 cell line provides an invaluable in vitro platform for mapping kinase pathway re-routing and developing combination targeted oncology regimens:

1. Targeted Therapeutics and Combination Screening for NRAS-Mutant Melanoma:

   While $BRAF^{V600E}$ melanomas respond highly to vemurafenib (PLX4032), these standard single-agent inhibitors are intrinsically ineffective against NRAS-driven M207 cells. Consequently, M207 serves as a benchmark model to evaluate novel Pan-RAF inhibitors combined with MEK inhibitors (e.g., TAK733, Trametinib), or to screen newly synthesized small-molecule ERK inhibitors to induce synthetic lethality.

2. Reverse-Phase Protein Array (RPPA) & Functional Proteomics:

   As a benchmark entity in the MD Anderson Melanoma Project database, M207 has deeply mapped Reverse-Phase Protein Array (RPPA) expression profiles. Investigators can retrieve calibrated quantitative data tracking hundreds of signaling hubs (such as p-ERK, p-Akt, p-S6) under baseline or drug-stressed states, making it ideal for systems biology modeling.

3. Tumor Invasion and Microenvironmental Mechanics:

   Driven by combined PTEN deficiency and oncogenic NRAS, this cell line exhibits high migratory, invasive, and extracellular matrix (ECM) penetrative capacities. It is routinely integrated into scratch wound-healing models, Transwell invasion assays, and studies decoding paracrine cross-talk between melanoma cells, endothelial sheets, and cancer-associated fibroblasts (CAFs).

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Complete Growth Medium

M207 adapts seamlessly to standard nutrient baselines. The optimized recipe includes:

• Basal Medium: RPMI 1640 Growth Medium (Highly Recommended) (pre-fortified with 2.0 mM L-Glutamine).

• Complete Supplements:

  • 10% Premium Heat-Inactivated Fetal Bovine Serum (FBS).

  • 1% Sterilized Penicillin-Streptomycin cocktail (100 U/mL final working threshold).

• Environment: 37°C under a humidified atmosphere containing 5% $CO_2$.

2. Cryovial Thawing Routine

1. Pre-warm the formulated complete growth medium inside a 37°C water bath.

2. Extract the M207 cryovial from liquid nitrogen and instantly submerge it into the 37°C water bath with continuous horizontal agitation.

3. Achieve complete liquefaction rapidly within 1 minute (remove when a minimal frozen core remains). Swab the exterior thoroughly with 75% ethanol before transferring it into the biosafety hood.

4. Pipette the cell suspension gently and drop it slowly into a 15 mL tube containing 5 mL of pre-warmed complete growth medium to mitigate sudden osmotic shock.

5. Centrifuge at 200–300 × g (~1000 rpm) for 3–4 minutes, then cleanly aspirate the DMSO-laden supernatant fluid.

6. Replenish with 5 mL of fresh complete medium, smoothly resuspend the pellet via gentle pipetting, and transfer into a T25 culture flask. Incubate at 37°C with 5% $CO_2$ and check attachment profiles the following day.

3. Subculturing and Cell Passaging Guide

• Confluency Cutoff: Due to its rapid doubling time (~25 hours), passaging must occur precisely when the monolayer reaches 70%–80% confluency. Avoid overgrowth, as extreme cell-packing and NRAS-driven metabolic shifts can trigger non-specific detachment or cell death.

• Split Ratios & Seeding Density: Split vessels utilizing standard split ratios of 1:3 to 1:5 every 2 to 3 days.

• Step-by-Step Harvesting Routine:

  1. Aspirate spent medium and rinse the cell layer 1–2 times with sterile, calcium/magnesium-free PBS to remove residual serum proteins that neutralize trypsin.

  2. Add an appropriate volume of 0.25% Trypsin-EDTA Solution (approx. 1 mL for standard T25 flasks) to submerge the monolayer.

  3. Incubate at 37°C for 1 to 3 minutes. Monitor continuously under an inverted microscope; as soon as the polygonal cell boundaries round up and detach upon gentle mechanical tapping, instantly add a double volume of serum-containing complete growth medium to quench enzymatic activity.

  4. Gently pipette the vessel walls to yield a homogeneous single-cell suspension. Centrifuge at 300 × g for 3 minutes, then discard the supernatant fluid.

  5. Disperse the pellet into fresh complete medium, split according to target ratios, and allocate into new culture vessels.

4. Cryopreservation Protocol

• Freezing Medium Matrix: 90% Complete Growth Medium (or pure premium FBS) supplemented with 10% analytical-grade cell-culture DMSO.

• Freezing Routine: Harvest active, log-phase cultures showing >90% cell viability metrics. Centrifuge and resuspend the cell mass to hit a final concentration baseline of $1.5 \times 10^6$ to $3 \times 10^6\text{ cells/vial}$. Aliquot into sterile cryovials and transfer immediately into a standardized controlled-rate freezing box (e.g., Mr. Frosty container, ensuring a constant thermal drop profile of -1°C/minute). House at -80°C overnight, and shift the vials into the liquid nitrogen vapor phase (-196°C) the following day for long-term preservation.

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